Method of reducing the harmful effects of orally or transdermally delivered nicotine

ABSTRACT

The present invention generally relates to the reduction of the harmful effects of orally or transdermally delivered nicotine in conventional tobacco-use cessation programs. More specifically, embodiments concern methods of reducing the harmful effects of nicotine intake, associated with conventional tobacco-use cessation programs, by providing tobacco products, which contain a reduced amount of nicotine and/or tobacco specific nitrosamines (TSNAs).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priority to U.S. patent application Ser. No. 11/293,680, filed on Dec. 2, 2005, which is a continuation-in-part of, and claims the benefit of priority to, international patent application number PCT/US2004/016958, filed May 27, 2004, which designated the United States of America and was published in English and which claims the benefit of priority to U.S. provisional patent application No. 60/475,945, filed Jun. 4, 2003; U.S. patent application Ser. No. 11/293,680 is also a continuation-in-part of and claims the benefit of priority to PCT/US2005/10733, filed Mar. 29, 2005, which designated the United States of America and was published in English and which claims the benefit of priority to 60/557,929, filed Mar. 30, 2004; U.S. patent application Ser. No. 11/293,680 is also a continuation-in-part of and claims the benefit of priority to U.S. patent application Ser. No. 11/077,752, filed Mar. 10, 2005, which is a continuation of U.S. patent application Ser. No. 10/729,121, filed Dec. 5, 2003, now U.S. Pat. No. 6,907,887, which is a continuation of PCT/US2002/18040, filed Jun. 6, 2002, which designated the United States of America and was published in English and which claims the benefit of priority to U.S. provisional application number 60/297,154, filed Jun. 8, 2001; and U.S. patent application Ser. No. 11/293,680 also claims the benefit of priority to U.S. provisional patent application No. 60/680,283, filed May 11, 2005. All of the aforementioned patent applications and provisional patent applications are hereby expressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

Aspects of the invention concern tobacco-use cessation programs that employ reduced nicotine tobacco products. More specifically, embodiments include methods of reducing nicotine and/or Tobacco Specific Nitrosamine (TSNA) consumption in a tobacco user by providing said tobacco user with a plurality of tobacco products, which contain a reduced amount of nicotine and/or TSNAs, preferably, in a step-wise manner that gradually reduces the exposure of the tobacco user to nicotine and/or TSNAs.

BACKGROUND OF THE INVENTION

The addictive properties of tobacco products are largely attributable to the presence of nicotine and the habitual use of the delivery system (e.g., the oral fixation associated with the act of smoking or chewing tobacco, smoke intake, and taste). Many tobacco-use cessation programs involve the use of nicotine replacement therapy (NRT) products, wherein various amounts of nicotine are given to the individual as a replacement for tobacco use. Several types of tobacco-use cessation products, which involve NRT, are currently available. For example, nicotine patches, gums, capsules, inhalers, nasal sprays, and lozenges are conventional products of NRT. Although these conventional products of NRT may help tobacco users by suppressing the symptoms of nicotine withdrawal, they do little to satisfy a tobacco user's cravings for the habitual use of the delivery system. (Dotinga, Study Bursts Nicotine Gum's Bubble, Health—Health Scout News, Sep. 20, 2002). The factors involved with the habitual use of the delivery system are hereinafter referred to as “secondary factors of addiction.” These secondary factors of addiction are largely psychological and have only an incidental relationship to the chemical dependence on nicotine.

In addition to the fact that conventional NRT does little to quell the secondary factors of addiction, NRT has had only limited success in enabling people to quit tobacco use. For example, among over-the-counter NRT gum users, abstinence rates were 16.1% at 6 weeks and 8.4% at 6 months; whereas, for prescription NRT gum users abstinence rates were 7.7% at 6 weeks and 7.7% at 6 months. (Shiffman et al., Addiction 97:505-516, 2002). Users of the of the NRT patch experienced only slightly better results; over-the counter patch users were reported to have 19.0% abstinence at 6 weeks and 9.2% at 6 months; whereas, prescription NRT patch users experienced 16.0% abstinence at 6 weeks and 3.0% abstinence at 6 months. Id. Others report slightly better results in that smoking cessation with patch or gum show verified abstinence rates at 12 months in the range of 20%. (O'Brien, Lecture given to medical students at the University of Pennsylvania on Sep. 22, 1995). One study, however, goes so far as to say that NRT is no longer effective in increasing long-term successful cessation in California smokers. (Pierce and Gilpin, Jama, 288:1260-1264 (2002)). Clearly, it appears that tobacco addiction is a complex web of psychological factors (i.e., the secondary factors) coupled with nicotine dependence and existing NRT is largely ineffective.

By design, conventional NRT relies on tobacco users to gradually reduce their daily nicotine intake, while they mentally curb their cravings for the secondary factors of addiction. In practice, however, many program participants only replace the addiction for tobacco with a far more expensive addiction to the NRT product. In some cases, program participants continue using the NRT product for long periods after the initial program has been completed and eventually return to tobacco products. There remains a need for tobacco-use cessation programs that focus on the secondary factors of addiction while reducing the harmful effects of nicotine.

SUMMARY OF THE INVENTION

Aspects of the present invention relate to methods of reducing nicotine dependence of a tobacco user by gradually reducing the exposure of said tobacco user to nicotine while maintaining use of a tobacco product. It should be appreciated that although TSNAs themselves may have some addictive properties, the primary addictive component in tobacco is nicotine. Accordingly, throughout this disclosure it is intended that embodiments directed to a reduction in nicotine dependence or consumption of a tobacco user are focused on a reduction of nicotine in tobacco products provided to said tobacco user while embodiments directed to a reduction in carcinogenic potential or consumption of TSNAs by a tobacco user are focused on a reduction in the presence of TSNAs in tobacco products that are provided to the tobacco user.

Tobacco products comprising a reduced amount of nicotine and/or TSNAs (e.g., N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK)) have been developed. These reduced nicotine and/or TSNA tobacco products have been provided to tobacco users in a step-wise program that gradually reduces the exposure of the tobacco user to nicotine and/or TSNAs. The nicotine and/or TSNA consumption of a tobacco user that follows the methods described herein is gradually reduced and, preferably, the consumption of nicotine of program participants is reduced to a level that allows the tobacco user to quit tobacco use entirely. Accordingly, aspects of the invention concern the tobacco products used in the tobacco-use cessation or nicotine and/or TSNA reduction programs described herein, kits that contain reduced nicotine and/or TSNA tobacco products, and methods of making and using these compositions so as to reduce the exposure of a tobacco user to nicotine and/or TSNAs, while maintaining use of a tobacco product (e.g., snuff chew, loose-leaf, or cigarettes).

The reduced nicotine and/or TSNA tobacco products described herein can be made by a variety of methods, which can be used separately or in combination. In general, these methods fall into three categories: “treated tobacco,” “selectively bred low nicotine tobacco,” or “genetically modified tobacco.” By “reduced nicotine and/or TSNA tobacco or tobacco product” is meant that the tobacco or tobacco product comprises an amount of nicotine and/or TSNAs that is less than that of the same variety of tobacco or tobacco product (e.g., full-flavor, light, or ultra-light) grown or prepared under similar conditions, wherein the tobacco and/or tobacco product has not been treated or modified to have a reduced amount of nicotine and/or TSNAs. In some embodiments, a wild-type tobacco of the variety that has been modified, which is grown, harvested, and cured under the about the same conditions, or a reference tobacco or tobacco product (e.g., 2R4F or IM-16) is used as the standard by which to determine the presence or absence of a particular modification that reduces the amount of nicotine and/or TSNAs. Accordingly, some embodiments described herein are not bound by the mechanism or approach used to create the reduced nicotine and/or TSNA tobacco.

In the United States, tar, nicotine, and carbon monoxide yields are obtained using the Federal Trade Commission (FTC) smoking-machine test method, which defines the measurement of tar as that material captured by a Cambridge pad when a cigarette is machine smoked, minus nicotine and water (Pillsbury, et al., 1969, “Tar and nicotine in cigarette smoke”. J. Assoc. Off. Analytical Chem., 52, 458-62). Specifically, the FTC cigarette-testing method collects smoke samples by simulating puffing volumes of 35 ml of cigarette smoke for two seconds every 58 seconds, with none of the filter ventilation holes blocked (if any), until the burn line reaches the tipping paper plus 2 mm, or a line drawn 23 mm from the end of a non-filter cigarette. This FTC smoking-machine test method has been used in the United States since 1967 to determine smoke cigarette yields for tar and nicotine. The determination of carbon monoxide yields in cigarette smoke was added to this method in 1980.

In 1967, when the FTC introduced its testing method, it issued a news release and explained that the purpose of the testing “is not to determine the amount of tar and nicotine inhaled by any human smoker, but rather to determine the amount of tar and nicotine generated when a cigarette is smoked by a machine in accordance with the prescribed method.” Nevertheless, the method serves an important role in providing an accurate way to rank and compare cigarettes according to tar, nicotine and carbon monoxide yields.

The International Standards Organization (ISO) developed a very similar smoking-machine test method for tar, nicotine, and carbon monoxide yields of cigarettes (ISO, 1991 “Cigarettes—determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine” ISO: 4387:1991).

The FTC and ISO smoking methods differ in the following eight areas.

-   -   The FTC method specifies laboratory environmental conditions of         75° F.±1° F. (23.8° C.±1° C.) and a relative humidity of 60%±2%         for both the equilibration and testing. The time of         equilibration is a minimum of 24 hours and a maximum of 14 days.         This is compared to the ISO specifications of 22° C.±1° C. and         60%±2% relative humidity for equilibration, 22° C.±2° C. and 60%         relative humidity±5% for testing. The equilibration time is a         minimum of 48 hours and a maximum of 10 days.     -   The FTC defines the cigarette butt length as a minimum of 23         millimeters or the tipping paper plus three millimeters         whichever is longer. ISO defines butt length as the longest of         23 millimeters or tipping paper plus three millimeters or the         filter plus eight millimeters. Both methods specify a         23-millimeter butt length for non-filter cigarettes.     -   ISO defines the position of the ashtray at 20-60 millimeters         below the cigarettes in the smoking machine. FTC does not         specify a position.     -   ISO specifies a two-piece snap together reusable filter holder.         This filter holder contains the Cambridge pad and uses a         synthetic rubber perforated washer to partly obstruct the butt         end of the cigarette. The FTC method defines the use of a         Cambridge filter pad but does not specify a filter pad holder         assembly.     -   The ISO method specifies airflow across the cigarettes at the         cigarette level. FTC specifies the use of a monitor cigarette to         adjust airflow.     -   The ISO procedure defines the process of wiping the excess total         particulate matter (TPM) out of the used filter holder. The         inner surfaces of the filter holder are wiped with two separate         quarters of an unused conditioned filter pad. The FTC method         uses the backside (the side opposite of the trapped TPM) to wipe         the inner surface of the filter holder.     -   ISO specifies using 20 ml per Cambridge pad of extraction         solution to analyze nicotine and water in TPM. The FTC procedure         defines 10 ml per Cambridge pad.     -   ISO defines the internal standards for the gas chromatographic         determination of nicotine and water. The FTC procedure does not         specify the internal standards.

These differences typically result in slightly lower measured deliveries for the ISO Method versus the FTC Method. The measured values between FTC and ISO methods are within the detection limits of the test or about no greater than 0.4 mg tar and about 0.04 mg nicotine for cigarettes that yield over about 10 mg.

Thus, it should also be mentioned that it is understood in the art (and as used in the present disclosure) that when a tobacco or a tobacco product is said to “comprise”, “have”, or “contain” a particular amount of nicotine and/or TSNA, the tobacco or tobacco product itself may comprise, have, or contain the recited amount of nicotine and/or TSNA (e.g., the amount of nicotine and/or TSNA present in the tobacco leaf or on the tobacco rod) or, in some contexts, the recited amount of nicotine and/or TSNAs is the amount of nicotine and/or TSNAs present in the mainstream or sidestream smoke obtained from the tobacco or tobacco product using the FTC and/or ISO methodologies.

In some embodiments, the reduced nicotine and/or TSNA is provided in a tobacco product as a neat formulation in that only one variety or type of reduced nicotine and/or TSNA tobacco is provided; whereas, in other embodiments, the tobacco product comprises a blend or mixture of tobacco varieties or types of reduced nicotine and/or TSNA tobaccos. Preferably, the tobacco products (e.g., a cigarette) that are used in the tobacco-use cessation or nicotine and/or TSNA reduction kits and the tobacco-use cessation or nicotine and/or TSNA reduction methods described herein comprise a tobacco that comprises (e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 2.0 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. Some embodiments are limited to whether the tobacco is treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco, however, many embodiments are not so limited. Some embodiments also include an amount of exogenous nicotine.

In some embodiments, the reduced nicotine and/or TSNA tobacco products, which can be incorporated into the tobacco-use cessation or nicotine and/or TSNA reduction kits and methods described herein, are made from tobacco that has been treated or otherwise modified to reduce the amount of nicotine and/or TSNAs. Examples of “treated tobacco” include tobacco that has been treated by microbial enzymatic degradation, chemical treatment, high pressure extraction, reconstitution, and/or flash curing methods. Examples of these methods can be found, for example, in: U.S. Pat. No. 4,557,280; U.S. Pat. No. 4,561,452; U.S. Pat. No. 4,848,373; U.S. Pat. No. 4.183,364; U.S. Pat. No. 4,215,706; U.S. Pat. No. 5,803,081; U.S. Pat. No. 6,202,649; U.S. Pat. No. 6,425,401; U.S. Pat. No. 5,713,376; U.S. Pat. No. 6,338,348; U.S. Pat. No. 6,834,654; U.S. patent application Ser. No. 10/943,346, U.S. patent application Ser. No. 10/719,295 and WO 05/018307, which designated the United States and was published in English. The aforementioned patents and patent applications are hereby expressly incorporated by reference in their entireties. The reduced nicotine and/or TSNA tobaccos described above can be blended with conventional tobacco and/or can include extenders, fillers, tobacco substitutes (e.g., tomato or rosemary powder), stems, and scrap tobacco and can contain exogenous nicotine. The treated tobacco that can be used in the methods described herein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 2.0 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. Similarly, tobacco products that comprise the treated tobacco above can be used in the tobacco-use cessation or nicotine and/or TSNA reduction kits and methods described herein. These tobacco products may comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g.

Desirably, the reduced nicotine and/or TSNA tobacco products, which can be incorporated into the tobacco-use cessation or nicotine and/or TSNA reduction kits and methods described herein, are made from “selectively bred low nicotine tobacco.” For example, some embodiments described herein comprise “selectively bred low nicotine tobacco,” such as low nicotine burley varieties (e.g., Burley 21 LA), low nicotine flue-cured varieties (e.g., NC-13 or LA FC53), low nicotine air-cured varieties (e.g., N506) or low nicotine oriental varieties (e.g., Nevrokop or Melnik varieties). Tobacco products comprising tobacco that has been selectively bred or grown to have a reduced amount of nicotine and/or TSNAs can also be blended with conventional tobacco and/or can include extenders, fillers, tobacco substitutes (e.g., tomato or rosemary powder), stems, and scrap tobacco and can contain exogenous nicotine. The selectively bred low nicotine tobacco that can be used in the methods described herein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 2.0 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. Similarly, tobacco products that comprise the selectively bred low nicotine tobacco above can be used in the tobacco-use cessation or nicotine and/or TSNA reduction kits and methods described herein. These tobacco products may comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g.

Preferably, the reduced nicotine and/or TSNA tobacco products, which can be incorporated into the tobacco-use cessation or nicotine and/or TSNA reduction kits and methods described herein, are made from tobacco that has been genetically modified to comprise a reduced amount of nicotine and/or TSNAs. In some embodiments, the “genetically modified” tobacco comprises an exogenous nucleic acid that encodes a protein in the nicotine biosynthesis pathway (e.g., A622, quinolate phosphoribosyl transferase (QPTase), putrescene N-methyl transferase (PMTase), N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase) or a fragment thereof. The exogenous nucleic acid that encodes a protein in the nicotine biosynthesis pathway or a fragment thereof (e.g., 25, 30, 50, 75, 100, 200, or 500 consecutive nucleotides in length) can be incorporated into a construct that inhibits the production of nicotine by many different modalities, depending on the construct (e.g., antisense, RNA interference, co-suppression, and/or molecular decoy), when said construct is introduced into a tobacco plant cell (e.g., by bacterial or biolistic transformation) and a transgenic plant is regenerated from the transformants. Exemplary methods for creating “genetically modified tobacco” include the approaches described in, for example, W098/56923; U.S. Pat. Nos.: 6,586,661; 6,423,520; 6,907,887. and U.S. patent application Ser. Nos. 09/963,340; 10/356,076; 09/941,042; 10/363,069; 10/729,121; 10/943,346; 11/077,752; WO00/67558, which designated the United States and was published in English; U.S. Pat. Nos.: 5,684,241; 5,369,023; 5,260,205; and 6,700,040, all of which are hereby expressly incorporated by reference in their entireties. Tobacco products comprising tobacco that has been genetically modified to have a reduced amount of nicotine and/or TSNAs can also be blended with conventional tobacco and/or can include extenders, fillers, tobacco substitutes (e.g., tomato or rosemary powder), stems, and scrap tobacco and can include exogenous nicotine. The genetically modified tobacco that can be used in the methods described herein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 2.0 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. Similarly, tobacco products that comprise the genetically modified tobacco above can be used in the tobacco-use cessation or nicotine and/or TSNA reduction kits and methods described herein. These tobacco products may comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g.

As described above, some of the tobaccos described herein contain an amount of exogenous nicotine. More specifically, some of the tobaccos described herein comprise an amount of exogenous nicotine (e.g., nicotine prepared by extraction of conventional tobacco or synthetically prepared nicotine) so as to adjust the content of the nicotine in the tobacco to a desirable level, which allows for fine adjustments in the amount of nicotine present in a tobacco product that comprises this tobacco and, accordingly, fine adjustments in the amount of nicotine provided to a tobacco user. Preferably, the tobacco is maintained in a microbe-free environment after addition of the exogenous nicotine so as to prevent the accumulation of TSNAs. Accordingly, the tobacco products described herein can include exogenous nicotine and the tobacco-use cessation kits and tobacco-use cessation methods described herein can include these exogenous nicotine-containing tobacco products.

Some embodiments concern methods of making the tobacco products described herein. By some approaches, a blended reduced nicotine tobacco is made by providing a first tobacco, which can be a conventional tobacco or a modified tobacco as described above (e.g., a treated tobacco, a selectively bred low nicotine tobacco, or a genetically modified tobacco); providing a second tobacco, which can be a conventional tobacco or a modified tobacco as described above (e.g., a treated tobacco, a low nicotine selectively bred tobacco, or a genetically modified tobacco); and blending the first and second tobaccos so as to obtain a reduced nicotine and/or TSNA blended tobacco. Tobacco products and tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise the blended reduced nicotine and/or TSNA tobacco produced by this method are also provided. The blended reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. The blended tobacco products can also include exogenous nicotine. The blended tobacco that can be used in the methods described herein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 2.0 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. Similarly, tobacco products that comprise the blended tobacco above can be used in the tobacco-use cessation or nicotine and/or TSNA reduction kits and methods described herein. These tobacco products may comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g.

Additional embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise one or more of the reduced nicotine and/or TSNA tobacco products described herein. Although some embodiments comprise a tobacco-use cessation or nicotine and/or TSNA reduction kit that comprises only one tobacco product that comprises a tobacco that has a reduced amount of nicotine and/or TSNAs, as compared to a conventional tobacco of the same variety grown under the same conditions; preferred embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise a plurality of tobacco products, wherein at least two of said tobacco products comprise different amounts of nicotine and/or TSNAs.

Some tobacco-use cessation or nicotine and/or TSNA reduction kits comprise, for example, a conventional tobacco product and a first reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the first tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the conventional tobacco product. The first reduced nicotine and/or TSNA tobacco product can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. The first tobacco product can also include exogenous nicotine.

Other embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise a conventional tobacco product, a first reduced nicotine and/or TSNA tobacco product and a second reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product and the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g and the second reduced nicotine and/or TSNA tobacco product can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the first tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the conventional tobacco product and the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product. The first and/or second reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine.

More embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise a conventional tobacco product, a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, and a third reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product, the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product and the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the third reduced nicotine and/or TSNA tobacco product can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the first tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the conventional tobacco product, the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product. The first, second, and/or third reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine.

Still more embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise a conventional tobacco product, a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, a third reduced nicotine and/or TSNA tobacco product and a fourth reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product, the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product, the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product, and the fourth reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the third reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μpg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the third reduced nicotine and/or TSNA tobacco product can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μpg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the fourth reduced nicotine and/or TSNA tobacco product can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the first tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the conventional tobacco product, the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the fourth tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the third tobacco product. The first, second, third, and/or fourth reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine.

Preferred tobacco-use cessation or nicotine and/or TSNA reduction kits comprise, however, a first reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than a conventional tobacco product. That is, in some embodiments, the tobacco-use cessation or nicotine and/or TSNA reduction kits do not contain a conventional tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. The first reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. The first tobacco product can also include exogenous nicotine.

Other embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise a first reduced nicotine and/or TSNA tobacco product and a second reduced nicotine and/or TSNA tobacco product, wherein the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g and the second reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product. The first and/or second reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine.

More embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, and a third reduced nicotine and/or TSNA tobacco product, wherein the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product and the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product or tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 ρg/g; and the third reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product. The first, second, and/or third reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine.

Still more embodiments include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, a third reduced nicotine and/or TSNA tobacco product and a fourth reduced nicotine and/or TSNA tobacco product, wherein the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product, the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product, and the fourth reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the third reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g. 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the third reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the fourth reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the fourth tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the third tobacco product. The first, second, third, and/or fourth reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine.

The tobacco-use cessation or nicotine and/or TSNA reduction kits described herein can, optionally, comprise instructions or guidance on use of the kit and/or tobacco-use cessation or nicotine and/or TSNA reduction and said instructions or guidance can refer the user to counseling programs and literature on the benefits of reduced exposure to nicotine and/or TSNAs and/or tobacco products, in general. The instructions or guidance can be provided in said kits in the form of a paper, CD-ROM, DVD, video, cassette, website link, or other tangible medium. Additionally, the tobacco products provided in said tobacco-use cessation or nicotine and/or TSNA reduction kits can also comprise indicia showing that the product is a member of a series of tobacco products to be consumed in a sequential order.

For example, in some embodiments, the tobacco products and/or packaging has been labeled with a number or letter or symbol or other form of visually identifiable marker to indicate whether the product is a conventional tobacco product, a first tobacco product, a second tobacco product, a third tobacco product, or a fourth tobacco product to be used in said kit or otherwise in conformance with a tobacco-use cessation or nicotine and/or TSNA reduction method described herein. Preferred indicia that identifies the tobacco product as a member of a series of tobacco products used in a tobacco-use cessation or nicotine and/or TSNA reduction method include visually identifiable rings or bars that appear on the tobacco product itself and/or the tobacco product packaging (see e.g., International Publication Number WO/05041151, which designates the U.S., and was published in English, herein expressly incorporated by reference in its entirety) and Quest 1®, Quest 2®, and Quest 3®. The tobacco-use cessation or nicotine and/or TSNA reduction kits and tobacco products and packing of such can also comprise indicia from a regulatory agency (e.g., a governmental body such as the Federal Drug Administration) indicating that said kit or the tobacco products contained therein have been approved for use in a tobacco-use cessation program.

Other embodiments concern methods of reducing the nicotine and/or TSNA consumption or exposure of a tobacco user by providing to said tobacco user a tobacco product or tobacco-use cessation or nicotine and/or TSNA reduction kit, as described herein. In some embodiments, a tobacco user is identified as one in need of a reduction in the consumption and/or exposure to nicotine and/or TSNAs. The identified tobacco user is then provided one or more of the aforementioned reduced nicotine and/or TSNA tobacco products and/or tobacco-use cessation kits described herein. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured.

Accordingly, by some approaches, a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs, is provided a conventional tobacco product and then said tobacco user is provided a first reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. The first reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. The first tobacco product can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

Other embodiments include tobacco-use cessation or nicotine and/or TSNA reduction methods, wherein a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs, is provided a conventional tobacco product and then said tobacco user is provided a conventional tobacco product, a first reduced nicotine and/or TSNA tobacco product and a second reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product and the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g and the second reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the first tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the conventional tobacco product and the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product. The first and/or second reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

More embodiments include tobacco-use cessation or nicotine and/or TSNA reduction methods, wherein a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs, is provided a conventional tobacco product, a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, and a third reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product, the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product and the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 g/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product or tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the third reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the first tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the conventional tobacco product, the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product. The first, second, and/or third reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

Still more embodiments include tobacco-use cessation or nicotine and/or TSNA reduction methods, wherein a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs, is provided a conventional tobacco product, a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, a third reduced nicotine and/or TSNA tobacco product and a fourth reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product, the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product, the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product, and the fourth reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the third reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the third reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the fourth reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the first tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the conventional tobacco product, the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the fourth tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the third tobacco product. The first, second, third and/or fourth reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

Preferred tobacco-use cessation or nicotine and/or TSNA reduction methods, however, include approaches, wherein a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs is provided a first reduced nicotine and/or TSNA tobacco product, wherein the first reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the conventional tobacco product. That is, said tobacco-use cessation or nicotine and/or TSNA reduction methods do not contain the step whereby a conventional tobacco product is provided. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g. The first reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. The first tobacco product can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

Other embodiments include tobacco-use cessation or nicotine and/or TSNA reduction methods, wherein a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs, is provided a first reduced nicotine and/or TSNA tobacco product and a second reduced nicotine and/or TSNA tobacco product, wherein the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g and the second reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product. The first and/or second reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

More embodiments include tobacco-use cessation or nicotine and/or TSNA reduction methods, wherein a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs, is provided a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, and a third reduced nicotine and/or TSNA tobacco product, wherein the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product and the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product or tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the third reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product. The first, second, and/or third reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

Still more embodiments include tobacco-use cessation or nicotine and/or TSNA reduction methods, wherein a tobacco user, who is, optionally, identified as one in need of a reduction in the consumption or exposure to nicotine and/or TSNAs, is provided a first reduced nicotine and/or TSNA tobacco product, a second reduced nicotine and/or TSNA tobacco product, a third reduced nicotine and/or TSNA tobacco product and a fourth reduced nicotine and/or TSNA tobacco product, wherein the second reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the first reduced nicotine and/or TSNA tobacco product, the third reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the second reduced nicotine and/or TSNA tobacco product, and the fourth reduced nicotine and/or TSNA tobacco product comprises less nicotine and/or TSNAs than the third reduced nicotine and/or TSNA tobacco product. The first reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the second reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; the third reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g; and the fourth reduced nicotine and/or TSNA tobacco product or a tobacco therein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g so long as the amount of nicotine and/or TSNAs in or delivered by the second tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the first tobacco product, the amount of nicotine and/or TSNAs in or delivered by the third tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the second tobacco product, and the amount of nicotine and/or TSNAs in or delivered by the fourth tobacco product is less than the amount of nicotine and/or TSNAs in or delivered by the third tobacco product. The first, second, third, and/or fourth reduced nicotine and/or TSNA tobacco products can comprise treated tobacco, selectively bred low nicotine tobacco, or genetically modified tobacco or combinations thereof. These tobacco products can also include exogenous nicotine. In some methods, the reduction in consumption or exposure to nicotine and/or TSNAs in said tobacco user is measured. In some methods, the abstinence from conventional tobacco use is measured. In some methods, a marker of nicotine addiction is measured (e.g., regional cerebral metabolic rate for glucose and/or cerebral blood flow, which are measurable using positron emission tomography (PET)).

In some embodiments, the tobacco-use cessation or nicotine and/or TSNA reduction kits and tobacco use cessation methods can also comprise a conventional NRT product (e.g., nicotine patches, nicotine gum, capsules, inhalers, nasal sprays, and lozenges). That is, aspects of the invention also include tobacco-use cessation or nicotine and/or TSNA reduction kits that comprise nicotine patches, nicotine gum, capsules, inhalers, nasal sprays, and lozenges that can be used in conjunction with a tobacco product as described herein. It is contemplated that the ability to quit tobacco use can be increased by providing a conventional NRT product in conjunction with one or more of the tobacco products described herein or supplementing one or more of the tobacco-use cessation methods described herein with a conventional NRT product and a conventional NRT nicotine-dependence reduction strategy. For example, a tobacco-use cessation or nicotine and/or TSNA reduction program can include the steps of providing a tobacco user who has, optionally, been identified as one in need of a reduction in conventional tobacco use one or more of the tobacco products described herein and a nicotine patch. Preferably, said tobacco user is provided a plurality of tobacco products described herein and a plurality of nicotine patches, wherein at least two tobacco products and at least two nicotine patches have different amounts of nicotine. That is, in some embodiments, a tobacco user is provided a first tobacco product that comprises a tobacco that has a reduced amount of nicotine (e.g., comprising on the leaf or tobacco rod or delivering in the side-stream or main-stream smoke, as determined by the FTC and/or ISO methods) less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g) and a nicotine patch comprising an amount of nicotine (e.g., 21 mg, 14 mg, or 7 mg).

In some embodiments, a tobacco user is provided at least two reduced nicotine tobacco products (e.g., a first tobacco product comprising on the leaf or tobacco rod or delivering in the side-stream or main-stream smoke, as determined by the FTC and/or ISO methods) less than or equal to 1.0 mg/g nicotine and a second tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.6 mg/g nicotine and a nicotine patch (e.g., 21 mg, 14 mg, or 7 mg nicotine); and, in other embodiments, a tobacco user is provided at least three reduced nicotine tobacco products described herein, for example, a first tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 1.0 mg/g nicotine, a second tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.6 mg/g nicotine, and a third reduced nicotine tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.3 mg/g nicotine) and a nicotine patch (e.g., 21 mg, 14 mg, or 7 mg nicotine); and, in some embodiments, a tobacco user is provided at least four tobacco products described herein, for example, a first tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 1.0 mg/g nicotine, a second tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.6 mg/g nicotine, a third reduced nicotine tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.3 mg/g nicotine, and a fourth reduced nicotine tobacco product comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.05 mg/g nicotine)and a nicotine patch (e.g., 21 mg, 14 mg, or 7 mg nicotine). Preferably, a tobacco user is provided a tobacco product that comprises (e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.05 mg/g nicotine and a nicotine patch comprising 21 mg, 14 mg, or 7 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. An illustration of a QPTase inhibition construct comprising a QPTase inhibition cassette including full-length QPTase coding sequence and a GUS selection cassette.

FIG. 2. An illustration of a QPTase inhibition construct comprising a QPTase inhibition cassette including a 360 bp fragment of the QPTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 3. An illustration of a PMTase inhibition construct comprising a PMTase inhibition cassette including a 241 bp fragment of the PMTase gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 4. An illustration of an A622 inhibition construct comprising an A622 inhibition cassette including a 628 bp fragment of the A622 gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

FIG. 5. An illustration of a QPTase/A622 double inhibition construct comprising a QPTase/A622 inhibition cassette including a 360 bp fragment of the QPTase gene and a 628 bp fragment of the A622 gene and a norflurazone resistance selection cassette including a mutant phytoene desaturase gene (PDSM-1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns nicotine reduction and/or tobacco-use cessation programs, which involve the use of modified tobacco products that contain reduced amounts of nicotine and/or TSNAs. While most tobacco cessation programs rely heavily on nicotine replacement therapy (NRT), many of the embodiments described herein focus less on nicotine replacement and more on replacing the secondary factors of addiction such as smoke intake, oral fixation, and taste. An application entitled “Modifying Nicotine and Nitrosamine Levels in Tobacco” (WO02100199), which was published in English designating the United States of America and claiming priority to U.S. Provisional Application No. 60/371635, is hereby incorporated by reference in its entirety. Also incorporated by reference in their entireties are related U.S. Pat. Nos. 6,586,661 and 6,423,520.

Some embodiments of the invention concern the use of low nicotine and/or TSNA tobacco products that have burning and taste characteristics that are virtually indistinguishable from conventional tobacco products. In some embodiments, taste characteristics are maintained by providing a tobacco product having an amount of tar similar to the amount of tar in standard tobacco products. That is, in some embodiments, the amount of tar in the various reduced nicotine and/or TSNA tobacco products (e.g., cigarettes comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine) is about the same even though the amount of nicotine in the tobacco products differs. The term “about” is used in this context and others to indicate that minor fluctuations in the levels of tar or other compounds are acceptable (e.g., in the context of tar, the term can signify that less than or equal to 1 mg, 0.7 mg, 0.5 mg, 0.25 mg, 0.1 mg, or 0.05 mg of tar higher or lower than a set value is acceptable). Thus, some embodiments described herein include tobacco products that comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) different levels of nicotine (e.g., less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine) and amounts of tar that are within 1 mg, 0.7 mg, 0.5 mg, 0.25 mg, 0.1 mg, or 0.05 mg higher or lower than a set value (e.g., 9 mg/g, 10 mg/g, 11 mg/g, or 12 mg/g) among different tobacco products. Tobacco use cessation programs that utilize these tobacco products are also embodiments.

While there are many ways to create reduced nicotine and/or TSNA products, the preferred methods use techniques in plant genetic engineering to reduce or eliminate enzymes involved in nicotine biosynthesis. Preferably, techniques in plant genetic engineering are used to selectively reduce the amount of an enzyme involved in nicotine biosynthesis, (e.g., the enzyme quinolate phosphoribosyl transferase (QPTase), which is involved in the production of nicotine at the root cortex). There may be many ways to reduce levels of QPTase in tobacco plants, given the teachings described herein and the level of skill in the art, however, the preferred methods involve the use of antisense, RNAi, cosuppression, or molecular decoy technology.

Several approaches to create tobacco and tobacco products that have a reduced amount of nicotine and/or TSNAs have been discovered. Interestingly, it was discovered that TSNA content in a tobacco plant can be lowered by reducing the nicotine content in the tobacco plant. In some embodiments, antisense technology is used to lower nicotine and TSNA levels in tobacco plants. (See PCT/US98/11893, which is hereby expressly incorporated by reference in its entirety). In other embodiments, molecular decoy technology is used to lower nicotine and/or TSNA levels in tobacco plants (See U.S. patent application Ser. No. 09/941,042, which is hereby expressly incorporated by reference in its entirety). In some embodiments, nucleic acid constructs encoding interfering RNAs (RNAi) comprising a first strand having a sequence substantially similar or identical to the entire coding sequence of a target gene and/or target gene product involved in nicotine biosynthesis, and a second strand that is complementary or substantially complementary to the first strand, are contemplated.

In some embodiments, the reduced nicotine tobacco products of the present invention are made utilizing tobacco that is treated to reduce its nicotine content after the tobacco has been harvested. Examples of such treatment include microbial enzymatic degradation, chemical treatment, high pressure extraction, reconstitution, and flash curing methods. (E.g., U.S. Pat. No. 4,557,280; U.S. Pat. No. 4,561,452; U.S. Pat. No. 4,848,373; U.S. Pat. No. 4,183,364; U.S. Pat. No. 4,215,706; U.S. Pat. No. 5,803,081; U.S. Pat. No. 6,202,649; U.S. Pat. No. 6,425,401; U.S. Pat. No. 5,713,376; U.S. Pat. No. 6,338,348; U.S. Pat. No. 6,834,654; U.S. patent application Ser. No. 10/943,346, and WO 05/018307). In other embodiments, the reduced nicotine and/or TSNA tobacco products are made from tobacco that has been selectively bred to reduce its nicotine and/or TSNA content. Accordingly, three general approaches to reducing nicotine and/or TSNAs are described: genetic modification, treatment (e.g., chemical or microbial), and selective breeding of low nicotine plants.

By a preferred approach, for example, a DNA construct encoding an antisense RNA that complements at least a portion of the QPTase gene (SEQ. ID. No. 1) is prepared such that transcription of the complementary strand of RNA reduces expression of the endogenous quinolate phosphoribosyl gene, which, in turn, reduces the amount of nicotine and, concomitantly, the amount of TSNA in the tobacco plant. By another approach, transcription factor molecular decoys for the QPTase gene, which are nucleic acid fragments that correspond to the 5′ upstream regulatory elements (e.g., Nic 1 and Nic 2 transcription factor binding sites) are inserted into the plant cell. The transcription factors bind to the decoy fragments rather than the endogenous transcription factor binding sites and a reduction in the level of transcription of QPTase is obtained.

Once the transgenic tobacco plants having reduced nicotine are made, the tobacco is harvested and cured by conventional methods and is incorporated into a variety of tobacco products. Preferably, the transgenic tobacco is blended such that specific amounts of nicotine and/or TSNA are obtained in specific products. That is, the blending is conducted so that tobacco products of varying amounts of nicotine and/or TSNAs are made. In this manner, a step-wise tobacco-use cessation program can be established, wherein a program participant begins the program at step 1 with a tobacco product having only slightly less nicotine than a conventional tobacco product; at step 2 the program participant begins using a tobacco product with less nicotine than the products used in step 1; and so on, for as many steps as desired for a particular tobacco-use cessation or nicotine and/or TSNA reduction program. Ultimately, the tobacco product used by the program participant can have an amount of nicotine that is less than that which is required to become addicted or maintain an addiction. In this manner, a nicotine and/or TSNA reduction and/or tobacco-use cessation program is provided that limits the exposure of a program participant to nicotine and/or TSNAs yet retains the secondary factors of addiction, including but not limited to, smoke intake, oral fixation, and taste. The following section describes tobacco products that can be used with the tobacco-use cessation programs described herein.

Tobacco Products for Use in Nicotine Reduction and/or Tobacco-Use Cessation Programs

Wild type tobacco varies significantly in the amount of TSNAs and nicotine depending on the variety and the manner it is grown, harvested, and cured. For example, a typical Burley tobacco leaf can have about 30,000 parts per million (ppm) nicotine and 8,000 parts per billion (ppb) TSNA; a typical Flue-Cured Burley leaf can have about 20,000 ppm nicotine and 300 ppb TSNA; and a typical Oriental cured leaf can have about 10,000 ppm nicotine and 100 ppb TSNA. A tobacco plant or portion thereof having a reduced amount of nicotine and/or TSNA, for use with aspects of the invention, can have no detectable nicotine and/or TSNA, or may contain some detectable amounts of one or more TSNA and/or nicotine so long as the amount of nicotine and/or TSNA is less than that found in a control plant of the same variety.

That is, a Burley tobacco leaf embodiment of the invention having a reduced amount of nicotine can have between about 0 and about 30,000 ppm nicotine and about 0 and about 8,000 ppb TSNA desirably, between about 0 and about 20,000 ppm nicotine and about 0 and about 6,000 ppb TSNA more desirably, between about 0 and about 10,000 ppm nicotine and about 0 and about 5,000 ppb TSNA preferably, between about 0 and about 5,000 ppm nicotine and about 0 and about 4,000 ppb TSNA more preferably, between about 0 and about 2,500 ppm nicotine and about 0 and about 2,000 ppb TSNA even more preferably, and most preferably between about 0 and about 1,000 ppm nicotine and about 0 and about 1,000 ppb TSNA. Embodiments of Burley leaf prepared by the methods described herein can also have between about 0 and about 1000 ppm nicotine and about 0 and about 500 ppb TSNA and some embodiments of Burley leaf prepared by the methods described herein have virtually no detectable amount of nicotine or TSNA.

Similarly, a Flue-cured tobacco leaf for use with the disclosed methods can have a reduced amount of nicotine, which is between about 0 and about 20,000 ppm nicotine and about 0 and about 300 ppb TSNA desirably between about 0 and about 15,000 ppm nicotine and about 0 and about 250 ppb TSNA more desirably between about 0 and about 10,000 ppm nicotine and about 0 and about 200 ppb TSNA preferably between about 0 and about 5,000 ppm nicotine and about 0 and about 150 ppb TSNA more preferably between about 0 and about 2,500 ppm nicotine and about 0 and about 100 ppb TSNA and most preferably between about 0 and about 1,000 ppm nicotine and about 0 and about 50 ppb TSNA. Embodiments of flue-cured tobacco prepared by the methods described herein can also have between about 0 and about 500 ppm nicotine and about 0 and about 25 ppb TSNA and some embodiments of flue-cured tobacco prepared by the methods described herein have virtually no detectable amount of nicotine or TSNA.

Further, an Oriental cured tobacco for use with the embodied methods can have a reduced amount of nicotine having between about 0 and about 10,000 ppm nicotine and about 0 and about 100 ppb TSNA desirably between about 0 and about 7,000 ppm nicotine and about 0 and about 75 ppb TSNA more desirably between about 0 and about 5,000 ppm nicotine and about 0 and about 50 ppb TSNA preferably between about 0 and about 3,000 ppm nicotine and about 0 and about 25 ppb TSNA more preferably between about 0 and about 1,500 ppm nicotine and about 0 and about 10 ppb TSNA and most preferably between about 0 and about 500 ppm nicotine and essentially no TSNA. Embodiments of Oriental cured tobacco prepared by the methods described herein can also have between about 0 and about 250 ppm nicotine and essentially no TSNA and some embodiments of Oriental cured tobacco prepared by the methods described herein have virtually no detectable amount of nicotine or TSNA.

As discussed above, TSNAs and nicotine contribute significantly to the carcinogenic potential and addictive properties of tobacco and tobacco products. Thus, tobacco and tobacco products that have a reduced amount of TSNA and nicotine have tremendous utility. It was found that the reduction of nicotine in tobacco was directly related to the reduction of TSNAs. Unexpectedly, the methods described herein not only produce tobacco with a reduced addictive potential but, concomitantly, produce a tobacco that has a lower carcinogenic potential.

It should be emphasized that the phrase “a reduced amount” is intended to refer to an amount of nicotine and/or TSNA in a tobacco plant, tobacco, or a tobacco product that is less than that found in a tobacco plant, tobacco, or a tobacco product from the same variety of tobacco grown and processed in the same manner, which has not been treated or was not made transgenic for reduced nicotine and/or TSNA. Thus, in some contexts, wild-type tobacco of the same variety that has been grown and processed in the same manner is used as a control by which to measure whether a reduction in nicotine and/or TSNA has been obtained.

In some contexts, the phrase reduced amount of nicotine and/or TSNAs refers to the tobacco plants, tobacco and tobacco products of the invention that have less nicotine and/or TSNAs by weight than the same variety of tobacco grown, processed, and cured in the same way. A typical cigarette has 11 mg of nicotine and 8 μg of TSNAs. Thus, the tobacco plants, tobacco and tobacco products of the invention can have, in dry weight for example, less than or equal to 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.35%, 0.375%, 0.4%, 0.425%, 0.45%, 0.475%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, and 1.0% nicotine and less than or equal to 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, and 0.08% TSNA.

Alternatively, a tobacco product, e.g., a cigarette, for use in the methods described herein can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg, 1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2.0 mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg, 2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg, 2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3.0 mg, 3.1 mg, 3.15 mg, 3.2 mg, 3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg, 3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, 4.0 mg, 4.1 mg, 4.15 mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4 mg, 4.45 mg, 4.4 mg, 4.45 mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg, 4.75 mg, 4.8 mg, 4.85 mg, 4.9 mg, 4.95 mg, 5.0 mg, 5.5 mg, 5.7 mg, 6.0 mg, 6.5 mg, 6.7 mg, 7.0 mg, 7.5 mg, 7.7 mg, 8.0 mg, 8.5 mg, 8.7 mg, 9.0 mg, 9.5 mg, 9.7 mg, 10.0 mg, 10.5 mg, 10.7 mg, 11.0 mg, 12.0 mg, 13.0 mg, 14.0 mg, 15.0 mg, 16.0 mg, 17.0 mg, 18.0 mg, 19.0 mg, or 20.0 mg nicotine and less than or equal to 0.1 micrograms, 0.15 micrograms, 0.2 micrograms, 0.25 micrograms, 0.3 micrograms, 0.35 micrograms, 0.4 micrograms, 0.45 micrograms, 0.5 micrograms, 0.55 micrograms, 0.6 micrograms, 0.65 micrograms, 0.7 micrograms, 0.75 micrograms, 0.8 micrograms, 0.85 micrograms, 0.9 micrograms, 0.95 micrograms, 1.0 micrograms, 1.1 micrograms, 1.15 micrograms, 1.2 micrograms, 1.25 micrograms, 1.3 micrograms, 1.35 micrograms, 1.4 micrograms, 1.45 micrograms, 1.5 micrograms, 1.55 micrograms, 1.6 micrograms, 1.65 micrograms, 1.7 micrograms, 1.75 micrograms, 1.8 micrograms, 1.85 micrograms, 1.9 micrograms, 1.95 micrograms, 2.0 micrograms, 2.1 micrograms, 2.15 micrograms, 2.2 micrograms, 2.3micrograms, 2.4 micrograms, 2.5 micrograms, 3.0 micrograms, 3.5 micrograms, 4.0 micrograms, 4.5 micrograms, or 5.0 micrograms TSNA (NNN, NNK, NAT, and NAB).

Moreover, the tobacco used in the claimed methods can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 0.01 mg/g, 0.05 mg/g, 0.1 mg/g, 0.15 mg/g, 0.2 mg/g, 0.25 mg/g, 0.3 mg/g, 0.35 mg/g, 0.4 mg/g, 0.45 mg/g, 0.5 mg/g, 0.55 mg/g, 0.6 mg/g, 0.65 mg/g, 0.7 mg/g, 0.75 mg/g, 0.8 mg/g, 0.85 mg/g, 0.9 mg/g, 0.95 mg/g, 1.0 mg/g, 1.1 mg/g, 1.15 mg/g, 1.2 mg/g, 1.25 mg/g, 1.3 mg/g, 1.35 mg/g, 1.4 mg/g, 1.45 mg/g, 1.5 mg/g, 1.55 mg/g, 1.6 mg/g, 1.65 mg/g, 1.7 mg/g, 1.75 mg/g, 1.8 mg/g, 1.85 mg/g, 1.9 mg/g, 1.95 mg/g, 2.0 mg/g, 2.1 mg/g, 2.15 mg/g, 2.2 mg/g. 2.25 mg/g, 2.3 mg/g, 2.35 mg/g, 2.4 mg/g, 2.45 mg/g, 2.5 mg/g, 2.55 mg/g, 2.6 mg/g, 2.65 mg/g, 2.7 mg/g, 2.75 mg/g, 2.8 mg/g, 2.85 mg/g, 2.9 mg/g, 2.95 mg/g, 3.0 mg/g, 3.1 mg/g, 3.15 mg/g, 3.2 mg/g, 3.25 mg/g, 3.3 mg/g, 3.35 mg/g, 3.4 mg/g, 3.45 mg/g, 3.5 mg/g, 3.55 mg/g, 3.6 mg/g, 3.65 mg/g, 3.7 mg/g, 3.75 mg/g, 3.8 mg/g, 3.85 mg/g, 3.9 mg/g, 3.95 mg/g, 4.0 mg/g, 4.1 mg/g, 4.15 mg/g, 4.2 mg/g, 4.25 mg/g, 4.3 mg/g, 4.35 mg/g, 4.4 mg/g, 4.45 mg/g, 4.4 mg/g, 4.45 mg/g, 4.5 mg/g, 4.55 mg/g, 4.6 mg/g, 4.65 mg/g, 4.7 mg/g, 4.75 mg/g, 4.8 mg/g, 4.85 mg/g, 4.9 mg/g, 4.95 mg/g, 5.0 mg/g, 5.5 mg/g, 5.7 mg/g, 6.0 mg/g, 6.5 mg/g, 6.7 mg/g, 7.0 mg/g, 7.5 mg/g, 7.7 mg/g, 8.0 mg/g, 8.5 mg/g, 8.7 mg/g, 9.0 mg/g, 9.5 mg/g, 9.7 mg/g, 10.0 mg/g, 10.5 mg/g, 10.7 mg/g, 11.0 mg/g, 12.0 mg/g, 13.0 mg/g, 14.0 mg/g, 15.0 mg/g, 16.0 mg/g, 17.0 mg/g, 18.0 mg/g, 19.0 mg/g, or 20.0 mg/g nicotine and less than 0.1 micrograms/g, 0.15 micrograms/g, 0.2 micrograms/g, 0.25 micrograms/g, 0.3 micrograms/g, 0.35 micrograms/g, 0.4 micrograms/g, 0.45 micrograms/g, 0.5 micrograms/g, 0.55 micrograms/g, 0.6 micrograms/g, 0.65 micrograms/g, 0.7 micrograms/g, 0.75 micrograms/g, 0.8 micrograms/g, 0.85 micrograms/g, 0.9 micrograms/g, 0.95 micrograms/g, 1.0 micrograms/g, 1.1 micrograms/g, 1.15 micrograms/g, 1.2 micrograms/g, 1.25 micrograms/g, 1.3 micrograms/g, 1.35 micrograms/g, 1.4 micrograms/g, 1.45 micrograms/g, 1.5 micrograms/g, 1.55 micrograms/g, 1.6 micrograms/g, 1.65 micrograms/g, 1.7 micrograms/g, 1.75 micrograms/g, 1.8 micrograms/g, 1.85 micrograms/g, 1.9 micrograms/g, 1.95 micrograms/g, 2.0 micrograms/g, 2.1 micrograms/g, 2.15 micrograms/g, 2.2 micrograms/g, 2.3 micrograms/g, 2.4 micrograms/g, 2.5 micrograms/g, 2.6 micrograms/g, 2.7 micrograms/g, 2.8 micrograms/g, 2.9 micrograms/g, 3.0 micrograms/g, 3.5 micrograms/g, 4.0 micrograms/g, 4.5 micrograms/g, or 5.0 micrograms/g TSNA (NNN, NNK, NAT, and NAB).

It should also be appreciated that in some embodiments, the tobacco products of the present invention comprise an amount of tar similar to the amount of tar in standard cigarettes. In such embodiments, the tobacco product (e.g. a cigarette) can have about 0.5 mg to about 30 mg of tar. Such a tobacco product comprises (e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, less than or equal to 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 m, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29 mg, 29.5 mg, or 30 mg of tar. Accordingly, some embodiments described herein include tobacco-use cessation kits and tobacco-use cessation methods that comprise a plurality of tobacco products, wherein at least two of said tobacco products comprise or deliver different amounts of nicotine but the same amounts of tar. For example, some embodiments include tobacco products that comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine, wherein each product comprises about 10 mg tar.

Several methods for reducing endogenous levels of nicotine and TSNAs in a tobacco plant have been discovered. These approaches can be used to create the tobacco products described herein. Tobacco plants having a reduced amount of nicotine and/or TSNAs that retain good smoking characteristics and taste, as manufactured by the methods described in the following section, can be used in the embodiments described herein.

Approaches to Make Tobacco Products Having Reduced Nicotine and/or TSNA Levels

There are many methods for reducing the amount of nicotine and/or TSNAs present in tobacco and these approaches can be divided into three general categories: genetic modification, selective breeding to obtain low nicotine plants, and treatment of tobacco (e.g., microbe or chemical). (See e.g., U.S. Pat. No. 4,557,280; U.S. Pat. No. 4,561,452; U.S. Pat. No. 4,848,373; U.S. Pat. No. 4,183,364; U.S. Pat. No. 4,215,706; U.S. Pat. No. 5,803,081; U.S. Pat. No. 6,202,649; U.S. Pat. No. 6,425,401; U.S. Pat. No. 5,713,376; U.S. Pat. No. 6,338,348; U.S. Pat. No. 6,834,654; U.S. patent application Ser. No. 10/943,346, and WO 05/018307). In some embodiments, for example, tobacco can be contacted with an oxidizing agent (e.g., hydrogen peroxide) such that nicotine is converted to nicotinic acid. Another example is contacting tobacco with a substrate (e.g., another tobacco) that has been contacted with a strong acid (e.g., hydrochloric acid) such that the nicotine is transferred from the tobacco of interest to the substrate. Yet another example involves treating tobacco with a hot solution of potassium metabisulfite and, subsequently, boiled in a solution of potassium sulfate and potassium nitrate. A final example is contacting an unharvested tobacco plant with auxin, an auxin analog, or a jasmonate antagonist.

Nicotine can also be extracted from tobacco utilizing high pressure extraction. For example, tobacco can be subjected to a mixture of nitrogen and carbon dioxide gases (with the nitrogen being 50-80% of the mixture) at a pressure of 250 to 600 bar at temperatures greater than 50° C. Under these conditions, nicotine is removed from the tobacco. The extracted nicotine can be used in some embodiements that comprise exogenous nicotine, as described herein.

Nicotine can also be removed using microbial enzymatic degradation. For example, tobacco can be contacted with a microorganism (e.g., Cellulomonas sp. grown in a nitrate-containing medium) that effectively degrades alkaloids (including nicotine), resulting in a tobacco with a reduced amount of nicotine and nitrates.

Flash curing methods can also be used to reduce the amount of TSNAs present in tobacco. For example, tobacco can be exposed to microwave radiation as part of the curing process. Another example is curing tobacco under conditions that prevent anaerobic conditions around the tobacco. A curing system utilizing a high airflow of heated air (e.g. 100°-250° F.) is believed to result in a tobacco having a lower amount of TSNAs.

In addition, reconstituted or expanded tobacco can be used to make reduced nicotine tobacco products. Reconstituted tobacco is made from tobacco dust and other tobacco scrap material. It is processed into a sheet and then cut into strips to resemble tobacco. Expanded tobacco is tobacco that has been processed through expansion of suitable gasses, leaving the tobacco “puffed” and therefore having a reduced density and greater filling capacity. Expanded tobacco can be used, for example, to reduce the weight of a cigarette which can reduced the amount of nicotine and/or TSNAs per cigarette.

Nicotine is produced in tobacco plants by the condensation of nicotinic acid and 4-methylaminobutanal. Two regulatory loci (Nic1 and Nic2) act as co-dominant regulators of nicotine production. These two loci are unlinked and the gene action is semi-dominant and primarily additive (Legg et al. (1 969) J. Hered., 60, 213-217).

Genetic and enzyme analyses have been used to investigate the Nic1 and Nic2 genes. Collins et al. ((1974) Crop Sci., 14, 77-80) prepared doubled haploid tobacco breeding lines of these four alkaloid genotypes. The genotype of standard cultivars is Nic1/Nic1 Nic2/Nic2 and that of low nicotine lines is nic1/nic1 nic2/nic2. Nic1/Nic1 nic2/nic2 is a high intermediate and nic1/nic1 Nic2/Nic2 is a low intermediate (Legg and Collins (1971) Can. J. Genet. Cytol. 13, 287-291). These lines are similar in days-to-flower, number of leaves, leaf size, and plant height. Enzyme analyses of roots of single and double Nic mutants show that the activities of two enzymes, quinolate phosphoribosyl transferase (QPTase) and putrescine methyl transferase (PMTase), are directly proportional to levels of nicotine biosynthesis (Saunders and Bush (1979) Plant Physiol 64:236). Both Nic1 and Nic2 affect PMTase and QPTase activities in roots, and thus, regulate nicotine synthesis (Leete (1983) In: Alkaloids: Chemical and Biological Perspectives, S. W. Pelletier, ed. John Wiley & Sons, pp. 85-152).

Enzyme analyses of roots of single and double Nic mutants show that the activities of QPTase and PMTase are directly proportional to levels of nicotine biosynthesis. An obligatory step in nicotine biosynthesis is the formation of nicotinic acid from quinolinic acid, which step is catalyzed by QPTase. QPTase appears to be a rate-limiting enzyme in the pathway supplying nicotinic acid for nicotine synthesis in tobacco (See, e.g., Feth et al., Planta, 168, pp. 402-07 (1986) and Wagner et al., Physiol. Plant., 68, pp. 667-72 (1986), herein expressly incorporated by reference in its entirety). A comparison of enzyme activity in tobacco tissues (root and callus) with different capacities for nicotine synthesis shows that QPTase activity is strictly correlated with nicotine content (Wagner and Wagner, Planta 165:532 (1985), herein expressly incorporated by reference in its entirety). In fact, Saunders and Bush (Plant Physiol 64:236 (1979), herein expressly incorporated by reference in its entirety, showed that the level of QPTase in the roots of low nicotine mutants is proportional to the levels of nicotine in the leaves.

Hibi et al. ((1994) Plant Cell, 6, 723-735) isolated the cDNA encoding PMTase, PMT, and showed that PMT transcript levels are regulated by Nic1 and Nic2. The QPTase cDNA and genomic clones (NtQPT1) have also been isolated and the transcript levels of NtQPT1 are also regulated by Nic1 and Nic2. Thus, it appears that the Nic genes regulate nicotine content by regulating the transcript levels of genes encoding the two rate-limiting enzymes, PMTase and QPTase. Further, Nic1 and Nic2 have been shown to be positive regulators of NtQPT1 transcription and that promoter sequences upstream of the transcription initiation site contain the cis-acting sequences necessary for Nic gene product activation of NtQPT1 transcription. Because expression of QPTase and PMTase are coordinately-regulated by the Nic gene products, it likely that the Nic gene products also directly regulate transcription of the PMT gene.

It has also been discovered that inhibition of A622 reduces the amount of nicotine in a tobacco plant. Accordingly, A622 encodes a gene product that regulates the production of nicotine. In some embodiments that employed the A622 inhibition construct described herein, transgenic tobacco that had conventional levels of nicotine but significantly reduced levels of nornicotine were produced. These lines of tobacco are particularly useful because nornicotine may be the most significant precursor for NNN in tobacco. Accordingly, reduced risk conventional cigarettes and other tobacco products (e.g., snuff) comprising the A622 inhibition construct are embodiments.

One approach for reducing nicotine involves reducing the amount of a required enzyme in the biosynthetic pathway leading to its production. Where the affected enzyme naturally occurs in a rate-limiting amount (relative to the other enzymes required in the pathway), any reduction in that enzyme's abundance will decrease the production of the end product. If the amount of the enzyme is not normally rate-limiting, its presence in a cell must be reduced to rate-limiting levels in order to diminish the pathway's output. Conversely, if the naturally-occurring amount of enzyme is rate limiting, then any increase in the enzyme's activity will result in an increase in the biosynthetic pathway's end product.

In some embodiments, the tobacco that is substantially free or comprises a reduced amount of nicotine, nornicotine, TSNAs, is made by exposing at least one tobacco cell of a selected variety (e.g., Burley, Virginia Flue, or Oriental) to an exogenous nucleic acid construct encoding an interfering RNA comprising an RNA duplex that comprises a first strand having a sequence that is substantially similar or identical to at least a portion of the coding sequence of a target gene and/or target gene product involved in nicotine biosynthesis, and a second strand that is complementary or substantially complementary to the first strand. In some embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding the interfering RNA operably linked to a promoter operable in a plant cell. The tobacco cell is transformed with the nucleic acid construct, transformed cells are selected and at least one transgenic tobacco plant is regenerated from the transformed cells. The transgenic tobacco plants described herein can contain a reduced amount of anyone of nicotine, nornicotine, and/or TSNAs, as compared to a control tobacco plant of the same variety. In some embodiments, nucleic acid constructs encoding interfering RNAs (RNAi) comprising a first strand having a sequence substantially similar or identical to the entire coding sequence of a target gene and/or target gene product involved in nicotine biosynthesis, and a second strand that is complementary or substantially complementary to the first strand, are contemplated.

Accordingly, some embodiments concern a tobacco that comprises a genetic modification comprising a reduced amount or a reduced level of expression of QPTase, PMTase, A622, or another enzyme in the plant's nicotine biosynthesis pathway, and/or comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) a reduced amount of nicotine or total alkaloid and/or a collective content of TSNA (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 2.0 μg/g (e.g., 2.0 μg/g, 1.75 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.2 μg/g). Other embodiments concern a tobacco that comprises a reduced amount or a reduced level of expression of A622, a normal or conventional amount of nicotine, for example, comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) equal to, less than, or greater than 0.9 mg/g, 1.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g, 1.6 mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, 2.0 mg/g, 3.0 mg/g, 4.0 mg/g, 5.0 mg/g, 6.0 mg/g, 7.0 mg/g, or 8.0 mg/g), and a reduced amount of nornicotine (e.g., less than or equal to 0.5 μg/g, 0.4 μg/g, 0.3 μg/g, 0.2 μg/g, or 0.1 μg/g and/or a reduced amount of NNN (e.g., less than or equal to 1.0 μg/g, 0.8 μg/g, 0.6 μg/g, 0.4 μg/g. 0.2 μg/g or 0.1 μg/g). That is, particular lines of transgenic tobacco containing the A622 inhibition cassette described herein were unexpectedly found to have a reduced level of nornicotine but conventional levels of nicotine. This finding is particularly important since nornicotine may be a more important precursor for NNN than nicotine. (See Carmella et al., Carcinogenesis, Vol. 21, No. 4, 839-843, (April 2000), herein expressly incorporated by reference in its entirety). In other transgenic lines, wherein the A622 gene was inhibited using one of the constructs described herein, it was found that both nicotine and nornicotine were effectively reduced (e.g., total alkaloids were less than or equal to 7,000 ppm, 5000 ppm, 3000 ppm, 1000 ppm, or 500 ppm).

In some embodiments, the gene product involved in nicotine biosynthesis is an enzyme. Such enzymes include, but are not necessarily limited to, putrescene N-methyltransferase (PMTase), N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase and quinolate phosphoribosyl transferase (QPTase). In preferred embodiments, the gene product that is inhibited using a construct described herein is QPTase and PMTase. In some embodiments, the tobacco that is made substantially free of nicotine and/or TSNAs (e.g., less than or equal to 0.5 mg/g nicotine and/or less than or equal to 0.5 μg/g collective content of NNN, NAT, NAB, and NNK) is prepared from a variety of Burley tobacco (e.g., Burley 21 LA), Oriental tobacco (Djebal 174), or Virginia flue (Tn90 or K326) tobacco. It should be understood, however, that most tobacco varieties (e.g., Burley, Flue, and Oriental) can be made to have reduced amounts of nicotine and/or TSNAs or can be made substantially free of nicotine and/or TSNAs by using the embodiments described herein. For example, plant cells of the variety Burley 21 LA are used as the host for the genetic engineering that results in the reduction of nicotine and/or TSNAs so that the resultant transgenic plants are a Burley 21 LA variety that has a reduced amount of nicotine and/or TSNAs.

Some of the nucleic acid constructs of the present invention employ interfering RNAs (e.g., siRNAs or dsRNAs) that comprise an RNA duplex wherein each RNA portion of the duplex is at least, greater than, or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 750, 1000, 1500, 2000, 2500, or 5000 consecutive nucleotides complementary or substantially complementary to an mRNA that encodes a gene product or the entire coding sequence of the enzyme or complement thereof of an enzyme that regulates nicotine biosynthesis. In some embodiments, the RNA duplex comprises a first RNA strand that is complementary to an mRNA that encodes a gene product involved in nicotine biosynthesis and a second RNA stand that is complementary to said first strand. Some interfering RNAs of the present invention can comprise two separate RNA strands hybridized to each other by hydrogen bonding. Other interfering RNAs comprise a single RNA strand comprising a first and second regions of nucleotide sequence that are complementary to each other. In such embodiments, the first and second regions of nucleotide sequence are separated by a nucleotide sequence (e.g., a “linker”) that permits or, in the case of the FAD2 intron described herein, facilitates formation of a hairpin structure upon hybridization of the first and second regions. This “linker” that permits formation of a hairpin structure is preferably at least, greater than, or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000 or more nucleotides in length.

The modification of nicotine levels in tobacco plants by antisense regulation of PMTase expression is proposed in U.S. Pat. Nos. 5,369,023 and 5,260,205 to Nakatani and Malik, all of which are hereby expressly incorporated by reference in their entireties. PCT application WO 94/28142 to Wahad and Malik describes DNA encoding PMT and the use of sense and antisense PMT constructs. Additionally, PCT Application WO98/56923 to Conkling et al. describes DNA encoding a plant QPTase enzyme, constructs comprising such DNA, and methods of altering QPTase expression to increase or decrease nicotine production in tobacco plants. Still further, U.S. patent application Ser. No. 09/941,042 to Conkling, which is hereby expressly incorporated by reference in its entirety, describes the use of DNA encoding regulatory sequences for the QPTase enzyme and methods of using these sequences as molecular decoys to sequester transcription factors at sites distant to the endogenous promoter for the QPTase gene, thereby decreasing nicotine production in tobacco plants.

Most notably, it is presently revealed that there are several different PMT genes and each may play a role in nicotine biosynthesis. Inhibition only one PMT gene may create a leaky system allowing the other genes to compensate for the reduction. Accordingly, the PMT constructs described herein were designed to inhibit a plurality of different PMT genes. That is, the PMT constructs described herein are designed to complement common regions to all five of the PMT genes so that inhibition of each of the PMT genes can be accomplished. Although many of the approaches described in this section have significant drawbacks, it should be understood that any or all of these techniques can be used with other techniques, as described herein, to make tobacco and tobacco products having reduced nicotine.

In some embodiments, the reduced nicotine tobacco products of the present invention are made utilizing tobacco that is treated to reduce its nicotine content after the tobacco has been harvested. Examples of such treatment include microbial enzymatic degradation, chemical treatment, or high pressure extraction. (See U.S. Pat. Nos. 4,557,280; 4,561,452; 4,848,373; 4,183,364; 4,215,706; and 5,713,376 all of which are hereby expressly incorporated by reference in their entireties). Tobacco products made from reconstituted tobacco are also usable in embodiments of the present invention, as described above.

Antisense Technology can be Used to Create Tobacco Products Having a Reduced Level of Nicotine and/or TSNA

Antisense technology may be used to create tobacco plants with reduced nicotine levels. The preferred enzyme for antisense regulation of nicotine levels is the TobRD2 gene (see Conkling et al., Plant Phys. 93, 1203 (1990)) encoding a Nicotiana tabacum QPTase (see Example 1) (SEQ. ID. No. 1). In addition to the description of the technology provided herein, general aspects of the technology are described in PCT/US98/11893, which is hereby expressly incorporated by reference in its entirety.

Regulation of gene expression in plant cell genomes can be achieved by integration of heterologous DNA under the transcriptional control of a promoter which is functional in the host, and in which the transcribed strand of heterologous DNA is complementary to the strand of DNA that is transcribed from the endogenous gene to be regulated. The introduced DNA, referred to as antisense DNA, provides an RNA sequence which is complementary to naturally produced (endogenous) mRNAs and which inhibits expression of the endogenous mRNA. Although the mechanism of antisense is not completely understood, it is known that antisense constructs can be used to regulate gene expression. A preferred approach for reducing QPTase levels through molecular modification is provided in Example 2 and Example 3.

In some methods, the antisense product may be complementary to coding or non-coding (or both) portions of naturally occurring target RNA. The antisense construct may be introduced into the plant cells in any suitable manner, and may be integrated into the plant genome for inducible or constitutive transcription of the antisense sequence. Tobacco plants are then regenerated from successfully transformed cells using conventional techniques. It is most preferred that the antisense sequence utilized be complementary to the endogenous sequence, however, minor variations in the exogenous and endogenous sequences may be tolerated. It is preferred that the antisense DNA sequence be of sufficient sequence similarity that it is capable of binding to the endogenous sequence in the cell to be regulated, under stringent conditions as described below.

Although the preferred enzyme for antisense regulation is QPTase, other enzymes that are suitable for antisense regulation include, for example, A622, putrescine N-methyltransferase, N-methylputrescine oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase, and any other enzyme linked to nicotine biosynthesis.

As an example of the use of antisense technology, tobacco having a reduced amount of nicotine and TSNA is generated from a tobacco plant that is created by exposing at least one tobacco cell of a selected tobacco variety (preferably Burley 21 LA) to an exogenous DNA construct having, in the 5′ to 3′ direction, a promoter operable in a plant cell and DNA containing a portion of a DNA sequence that encodes an enzyme in the nicotine synthesis pathway or a complement thereof (e.g., SEQ. ID. No. 1). The DNA is operably associated with said promoter and the tobacco cell is transformed with the DNA construct. The transformed cells are selected using either negative selection or positive selection techniques and at least one tobacco plant is regenerated from transformed cells. The regenerated tobacco plant or portion thereof is preferably analyzed to determine the amount of nicotine and/or TSNA present and these values can be compared to the amount of nicotine and/or TSNA present in a control tobacco plant or portion, preferably of the same variety.

The DNA constructs having a portion of a DNA sequence that encodes an enzyme in the nicotine synthesis pathway may have the entire coding sequence of the enzyme a complement of this sequence, or any portion thereof. A portion of a DNA sequence that encodes an enzyme in the nicotine synthesis pathway or the complement thereof may have at least 25, 27, 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, 1000, 1500, 2000, 2500, or 5000 bases, or the entire coding sequence of the enzyme or complement thereof (e.g., SEQ. ID. No. 1). Accordingly, these DNA constructs have the ability to inhibit the production of endogenous enzyme in the nicotine biosynthesis pathway. It is contemplated that antisense, molecular decoy, RNAi, and cosuppression constructs are effective at reducing the levels of nicotine and/or TSNAs in tobacco plants.

Nucleic acid sequences employed in the constructs described herein include those with sequence similarity to the gene encoding QPTase, and encoding a protein having quinolate phosphoribosyl transferase activity, including, for example, allelic variations in QPTase proteins. Thus, DNA sequences that hybridize to DNA of the QPTase-encoding gene and code for expression of QPTase, particularly plant QPTase enzymes, may also be employed in carrying out the present invention. Multiple forms of tobacco QPT enzyme may exist. Multiple forms of an enzyme may be due to post-translational modification of a single gene product, or to multiple forms of the NtQPT1 gene.

As used herein, the term ‘gene’ can refer to a DNA sequence that incorporates (1) upstream (5′) regulatory signals including the promoter, (2) a coding region specifying the product, protein or RNA of the gene, (3) downstream regions including transcription termination and polyadenylation signals and (4) associated sequences required for efficient and specific expression. In some contexts, a gene can include only (2), above, or some combination of items (1), (3), and (4) with (2). The DNA sequence of the present invention may comprise or consist essentially of the sequence encoding the QPTase enzyme, or equivalent nucleotide sequences representing alleles or polymorphic variants of these genes, or coding regions thereof. Use of the phrase “substantial sequence similarity” in the present specification and claims means that DNA, RNA or amino acid sequences which have slight and non-consequential sequence variations from the actual sequences disclosed and claimed herein are considered to be equivalent to the sequences of the present invention. In this regard, “slight and non-consequential sequence variations” mean that “similar” sequences (i.e., the sequences that have substantial sequence similarity with the DNA, RNA, or proteins disclosed and claimed herein) will be functionally equivalent to the sequences disclosed and claimed in the present invention. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein.

By one approach, a novel cDNA sequence encoding a plant QPTase may be used. As QPTase activity is strictly correlated with nicotine content, construction of transgenic tobacco plants in which QPTase levels are lowered in the plant roots (compared to levels in wild-type plants) result in plants having reduced levels of nicotine in the leaves. Embodiments of the invention provide methods and nucleic acid constructs for producing such transgenic plants, as well as the transgenic plants themselves. Such methods include the expression of antisense NtQPT1 RNA, which lowers the amount of QPTase in tobacco roots.

Aspects of the present invention also concern sense and antisense recombinant DNA molecules encoding QPTase or QPTase antisense RNA molecules, and vectors comprising those recombinant DNA molecules, as well as transgenic plant cells and plants transformed with those DNA molecules and vectors. Transgenic tobacco cells and the plants described herein are characterized in that they have a reduced amount of nicotine, as compared to unmodified or control tobacco cells and plants.

Promoters to be linked to the antisense constructs of the present invention may be constitutively active promoters. Numerous constitutively active promoters which are operable in plants are available. A preferred example is the Cauliflower Mosaic Virus (CaMV) 35S promoter which is expressed constitutively in most plant tissues. In the alternative, the promoter may be a root-specific promoter or root cortex specific promoter and others, as explained in greater detail below.

Antisense sequences have been expressed in transgenic tobacco plants utilizing the Cauliflower Mosaic Virus (CaMV) 35S promoter. See, e.g., Cornelissen et al., “Both RNA Level and Translation Efficiency are Reduced by Anti-Sense RNA in Transgenic Tobacco”, Nucleic Acids Res. 17, pp. 833-43 (1989); Rezaian et al., “Anti-Sense RNAs of Cucumber Mosaic Virus in Transgenic Plants Assessed for Control of the Virus”, Plant Mol. Biol. 11, pp. 463-71 (1988); Rodermel et al., “Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxylase Enzyme Levels in Transformed Tobacco Plants”, Cell 55, pp. 673-81 (1988); Smith et al., “Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes”, Nature 334, pp. 724-26 (1988); Van der Krol et al., “An Anti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation”, Nature 333, pp. 866-69 (1988).

Use of the CaMV 35S promoter for expression of antisense QPTase genes in the transformed tobacco cells and plants of this invention is preferred. Use of the CaMV promoter for expression of other recombinant genes in tobacco roots has been well described (Lam et al., “Site-Specific Mutations Alter In Vitro Factor Binding and Change Promoter Expression Pattern in Transgenic Plants”, Proc. Nat. Acad Sci. USA 86, pp. 7890-94 (1989); Poulsen et al. “Dissection of 5′ Upstream Sequences for Selective Expression of the Nicotiana plumbaginifolia rbcS-8B Gene”, Mol. Gen. Genet. 214, pp. 16-23 (1988).

Other promoters, which are active only in root tissues (root specific promoters) are also particularly suited to the methods of the present invention. (See, e.g., U.S. Pat. No. 5,459,252 to Conkling et al.; Yamamoto et al., Plant Cell, 3:371 (1991)). The TobRD2 root-cortex specific promoter may also be utilized. (See, e.g., U.S. patent application Ser. No. 08/508,786 and PCT WO 9705261, hereby expressly incorporated by reference n their entireties.

Some of the nucleic acids described herein may also be used in methods of sense co-suppression or RNAi-mediated suppression of nicotine production. Sense DNAs employed in these methods are preferably of a length sufficient to, when expressed in a plant cell, suppress the native expression of the plant QPTase protein as described herein in that plant cell. Such sense DNAs may be essentially an entire genomic or complementary DNA encoding the QPTase enzyme, or a fragment thereof, with such fragments typically being at least 15, 25, 27, 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, nucleotides in length. Methods of ascertaining the length of sense DNA that results in suppression of the expression of a native gene in a cell are available to those skilled in the art.

In an alternate embodiment, Nicotiana plant cells are transformed with a DNA construct containing a DNA segment encoding an enzymatic RNA molecule termed a “ribozyme”, which enzymatic RNA molecule is directed against and cleaves the mRNA transcript of DNA encoding plant QPTase as described herein. Production of such an enzymatic RNA molecule in a plant cell and disruption of QPTase protein production reduces QPTase activity in plant cells in essentially the same manner as production of an antisense RNA molecule: that is, by disrupting translation of mRNA in the cell which produces the enzyme. The section below describes yet another method to decrease levels of specific enzymes involved in nicotine biosynthesis, using decoy nucleic acid fragments.

Molecular Decoy Technology to Lower Nicotine and/or TSNA Levels

The use of nucleic acid-based decoy fragments to reduce gene expression is referred to as “molecular decoys”. In a preferred example, the “decoy fragment” corresponds to promoter sequences upstream of the QPTase gene, to reduce QPTase expression. In other embodiments, the “decoy fragment” corresponds to promoter sequences upstream of a gene encoding an enzyme involved in the plant's nicotine biosynthesis pathway (e.g., A622, QPTase, PMTase, N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase), that reduce the expression of the given gene.

In some embodiments, an isolated nucleic acid, or a fragment thereof consisting of at least 20-450 consecutive nucleotides desirably, at least 30-400 consecutive nucleotides preferably, 50-350 consecutive nucleotides more preferably, and 100-300 or 200-400 consecutive nucleotides most preferably, that is or contains at least one cis-acting regulatory element, which exists upstream of the plant QPTase (e.g., SEQ. ID. No. 1) and/or PMTase coding sequences. Another example is the Nic gene product responsive element obtained from the sequence disclosed in U.S. Pat. No. 5,459,252, herein expressly incorporated by reference in its entirety. In some embodiments, the Nic gene product responsive element resides between −1000 and −600 or −700 bp of the NtQPT1 promoter. Accordingly, some embodiments involve a 300-400 nucleotide long fragment of the NtQPT1 promoter that corresponds to the sequence of the NtQPT1 promoter between −1000 and −600 or −700, as disclosed in U.S. Pat. No. 5,459,252, herein expressly incorporated by reference in its entirety.

Thus, in several embodiments, the embodied nucleic acids have a structure that promotes an interaction with one or more transcription factors (e.g., Nic1 and Nic2), which are involved in initiating transcription of QPTase and/or PMTase. Accordingly, said nucleic acids are said to be or contain at least one transcription factor (e.g., Nic1 and Nic2) binding sequences, which are also referred to as “cis-acting regulatory elements.” By introducing multiple copies of these cis-acting regulatory elements (e.g., sequences that interact with Nic1 and/or Nic2) into a plant cell, the ability of the transcription factor to initiate transcription of the targeted gene (e.g., QPTase and/or PMTase genes) can be reduced or squelched.

By one approach, tobacco plants are transformed with an excess number of DNA sequences (cis-acting elements) from the promoters of genes encoding, but not limited to, QPTase and PMTase that are regulated in nicotine biosynthesis. These cis-acting elements are preferably integrated into the plant genome so as to allow for transfer to successive generations. Preferred approaches are provided in Example 4 and Example 5. Typically, the Nic1 and Nic2 DNA-binding proteins that interact with these cis-acting DNA sequences are expressed at relatively low levels in the cell, thus the excess of transgenic cis-acting elements will compete with the endogenous elements associated with the genes encoding, but not limited to, QPTase and PMTase for available Nic1 and Nic2 Accordingly, these cis-acting DNA sequences (and those of other cis-acting elements) are referred to herein as “decoys” or “molecular decoys”. The competition decreases occupancy of trans-acting DNA-binding proteins on their cognate cis-acting elements, thereby down-regulating the synthesis of nicotine biosynthesis enzymes.

Embodiments of the present invention also provide DNA molecules of cis-acting elements of QPTase or PMTase, and vectors comprising those DNA molecules, as well as transgenic plant cells and plants transformed with those DNA molecules and vectors. Transgenic tobacco cells and plants of this invention are characterized by lower nicotine content than untransformed control tobacco cells and plants.

Any of a variety of cis-acting elements can be used in carrying out the molecular decoy methods, depending upon the particular application. Examples of cis-acting elements (and corresponding transcription factors) that may be used, alone or in combination with one another, which may be used in embodiments of the present invention include, but are not limited to, AS-1 and ASF-1 (see U.S. Pat. Nos. 4,990,607 and 5,223,419), the AATT repeat element and PABF (see U.S. Pat. Nos. 5,834,236 and 6,191,258), a wounding-responsive cis-acting element from potato (Siebert et al., Plant Cell 1:961-8 (1989)), an embryo-specific cis-acting element from bean (Bustos et al, Plant Cell 1:839-853 (1989)), a root-specific cis-acting element from the tobacco RB7 promoter (U.S. Pat. No. 5,459,252 and Yamamoto et al., Plant Cell 3:371-382 (1991)), a positive poly(dA-dT) regulatory element and binding protein and negative CCCAA repeat element and binding protein (Wang et al., Mol. Cell Biol. 12:3399-3406 (1992)), a root-tip regulatory element from the tobacco phytochrome A1 promoter of tobacco (Adam et al., Plant Mol Biol 29:983-993 (1995)), an anaerobiosis-responsive element from the maize glyceraldehyde-3-phosphate dehydrogenase 4 gene (Geffers et al., Plant Mol Biol 43:11-21 (2000)), and a seed-specific regulatory region from an Arabidopsis oleosin gene (see U.S. Pat. No. 5,792,922), all of which are hereby expressly incorporated by reference in their entireties.

The status of the art is such that large databases list identified cis-acting regulatory regions (e.g., Plant Cis-acting Regulatory elements, “PLACE”, with about 1,340 entries, and Plant Cis-acting Regulatory Elements “PlantCARE”, which lists about 159 plant promoters. The listed cis-acting regulatory elements in these databases and the cis-acting regulatory elements that are provided in Raumbauts et al., Nucleic acids Research 27:295-296 (1999), and Higo et al., Nucleic acids Research 27:297-300 (1999) can be used with embodiments of the invention. Accordingly, the databases and references above are hereby expressly incorporated by reference in their entireties. The section below describes general methods for transformation of tobacco plants with modified sequences (RNAi) to create tobacco plants with low nicotine and/or TSNA levels.

Inhibition of Gene Expression Using RNAi

Inhibition of gene expression refers to the absence or observable reduction in the level of polypeptide and/or mRNA gene product. Some embodiments of the present invention relate to inhibiting the expression of one or more genes involved in the biosynthesis of nicotine and/or nornicotine by genetically modifying a plant cell, such as a tobacco cell, by providing the cell with an inhibitory nucleic acid that reduces or eliminates the production of a gene product involved in nicotine biosynthesis. Inhibitory nucleic acids include, but are not limited to, interfering RNAs, antisense nucleic acids and catalytic RNAs. Some preferred embodiments of the present invention relate to interfering RNAs (RNAi).

Target genes that are involved in nicotine and/or nornicotine biosynthesis are expressed through the transcription a first gene product, the target mRNA, which is then translated to produce a second gene product, the target polypeptide. Thus, reduction or elimination of the expression of one or more target genes results in the reduction or elimination of one or more target mRNAs and/or target polypeptides. Target polypeptides involved in nicotine and nornicotine biosynthesis include, for example, A622, PMTase, N-methylputrescene oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate isomerase, and QPTase. In a preferred embodiment, the expression of the QPTase, PMTase, and A622 product is inhibited using an RNAi construct provided herein.

Reduction of the expression of one or more target genes and/or target gene products that are involved in nicotine and/or nornicotine biosynthesis leads to a reduction in the amount of nicotine and/or TSNAs produced in tobacco. In certain embodiments, the expression of one or more target gene products involved in nicotine and/or nornicotine biosynthesis is eliminated. Elimination of such target gene products can result in the elimination of nicotine and/or nornicotine biosynthesis, thereby reducing the amount of nicotine and/or nornicotine present in tobacco to levels below the detection limit of methods commonly used. Reduction of the amount of nicotine and/or nornicotine present in tobacco can lead to a reduction in the amount of TSNAs produced in the tobacco. In some embodiments, the amount of TSNA present in tobacco is reduced to levels below the detection limit of methods commonly used to detect TSNAs.

The reduction in or elimination of the expression of target genes or target gene products involved in nicotine and/or nornicotine biosynthesis is achieved by providing an interfering RNA specific to one or more such target genes to a tobacco cell, thereby producing a genetically modified tobacco cell. The interfering RNA can be provided as a synthetic double-stranded RNA, or alternatively, as a nucleic acid construct capable of encoding the interfering RNA. Synthetic double-stranded interfering RNAs are taken up by the cell directly whereas interfering RNAs encoded by a nucleic acid construct are expressed from the construct subsequent to the entry of the construct inside the cell. The reduction in or elimination of the expression of the target genes and/or the target gene products is mediated by the presence of the interfering RNA inside the cell.

RNA interference and gene silencing are terms that are used to describe a phenomenon by which the expression of a gene product is inhibited by an interfering RNA molecule. Interfering RNA molecules are double-stranded RNAs (dsRNA) that are expressed in or otherwise introduced into a cell. The dsRNA molecules may by of any length, however, short dsRNA constructs are commonly used. Such constructs are known as small interfering RNAs (siRNA), and can be 21-23 bp in length.

RNA interference is exhibited by nearly every eukaryote and is thought to function by a highly conserved mechanism (Dillin, A. PNAS, 100:6289-91). As with antisense inhibition of gene expression, inhibition mediated by RNA interference is gene specific. However, in contrast to antisense-mediated inhibition, inhibition mediated by interfering RNA appears to be inherited (Dillin, A. PNAS, 100:6289-91). Without being bound by theory, it is believed that specificity is achieved through nucleotide sequence interaction between complementary portions of a target mRNA and the interfering RNA. The target mRNA is selected based on the specific gene to be silenced. In particular the target mRNA, corresponds to the sense strand of the gene to be silenced. An interfering RNA, such as a dsRNA or an siRNA, comprises an RNA duplex, which includes a first strand that is substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA, and a second strand having a nucleotide sequence that is complementary or substantially complementary to the first strand.

When used herein with reference to an RNA duplex of the interfering RNA, it will be appreciated that the terms “first strand” and “second strand” are used in a relative sense. For example, the first strand of an RNA duplex can be selected to comprise either a nucleotide sequence substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA or a nucleotide sequence that is complementary or substantially complementary to at least a portion of the nucleotide sequence of the target mRNA. If the first strand is selected to be substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA, then the second strand will be complementary to at least a portion of the target mRNA because it is complementary to the first strand. If the first strand is selected to be complementary or substantially complementary to at least a portion of the target mRNA, then the second strand will be substantially similar or identical to at least a portion of the nucleotide sequence of the target mRNA because it is complementary to the first strand.

In general, the interfering RNAs that are produced inside the cell, whether expressed from a nucleic acid construct or provided as synthetic double-stranded RNA molecules, include an RNA duplex having a first and second strand. At least a portion the first strand of the duplex is substantially similar or identical to at least a portion of a target mRNA or a target gene involved in nicotine biosynthesis. Correspondingly, at least a portion of the second strand of the duplex is complementary or substantially complementary to the first strand, and thus, at least a portion of the second strand is complementary or substantially complementary to at least a portion of the mRNA encoded by the target gene. In some embodiments, the interfering RNA can comprise a first strand that is substantially similar or identical to the entire coding sequence of the target gene or target mRNA involved in nicotine biosynthesis and a second strand complementary or substantially complementary to the first strand.

As used herein with reference to nucleic acids, “portion” means at least 5 consecutive nucleotides, at least 6 consecutive nucleotides, at least 7 consecutive nucleotides, at least 8 consecutive nucleotides, at least 9 consecutive nucleotides, at least 10 consecutive nucleotides, at least 11 consecutive nucleotides, at least 12 consecutive nucleotides, at least 13 consecutive nucleotides, at least 14 consecutive nucleotides, at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides, at least 21 consecutive nucleotides, at least 22 consecutive nucleotides, at least 23 consecutive nucleotides, at least 24 consecutive nucleotides, at least 25 consecutive nucleotides, at least 30 consecutive nucleotides, at least 35 consecutive nucleotides, at least 40 consecutive nucleotides, at least 45 consecutive nucleotides, at least 50 consecutive nucleotides, at least 60 consecutive nucleotides, at least 70 consecutive nucleotides, at least 80 consecutive nucleotides, at least 90 consecutive nucleotides, at least 100 consecutive nucleotides, at least 125 consecutive nucleotides, at least 150 consecutive nucleotides, at least 175 consecutive nucleotides, at least 200 consecutive nucleotides, at least 250 consecutive nucleotides, at least 300 consecutive nucleotides, at least 350 consecutive nucleotides, at least 400 consecutive nucleotides, at least 450 consecutive nucleotides, at least 500 consecutive nucleotides, at least 600 consecutive nucleotides, at least 700 consecutive nucleotides, at least 800 consecutive nucleotides, at least 900 consecutive nucleotides, at least 1000 consecutive nucleotides, at least 1200 consecutive nucleotides, at least 1400 consecutive nucleotides, at least 1600 consecutive nucleotides, at least 1800 consecutive nucleotides, at least 2000 consecutive nucleotides, at least 2500 consecutive nucleotides, at least 3000 consecutive nucleotides, at least 4000 consecutive nucleotides, at least 5000 consecutive nucleotides or greater than at least 5000 consecutive nucleotides. In some preferred embodiments, a portion of a nucleotide sequence is between 20 and 25 consecutive nucleotides. In other preferred embodiments, a portion of a nucleotide sequence is between 21 and 23 consecutive nucleotides. In some embodiments a portion of a nucleotide sequence includes the full-length coding sequence of the gene or the target mRNA.

Some preferred interfering RNAs that are described herein comprise an RNA duplex, which comprises a nucleotide sequence that is substantially similar or identical to at least a portion of the coding strand of a gene involved in nicotine biosynthesis. Although nucleic acid sequences that are substantially similar or identical to at least a portion of the coding strand of the target gene involved in nicotine biosynthesis are preferred, it will be appreciated that nucleotide sequences with insertions, deletions, and single point mutations relative to the target sequence are also effective for inhibition of gene expression. Sequence identity may determined by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the interfering RNA and a portion of the target gene is preferred. In especially preferred embodiments, at least about 21 to about 23 contiguous nucleotides in the target gene are greater than 90% identical to a sequence present in the interfering RNA.

In other embodiments, the duplex region of the RNA may be defined functionally as including a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. Exemplary hybridization conditions are 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing.

As described above, interfering RNAs disclosed herein comprise a sequence that is complementary to at least a portion of the sense strand of a gene encoding a target mRNA, which produces a polypeptide that is involved in nicotine biosynthesis. Preferred targets are the products of the A622 gene, quinolate phosphoribosyltransferase (QPTase) gene and the putrescene N-methyltransferase (PMTase) gene. However, it will be appreciated that interfering RNAs specific for other gene products or combinations of gene products involved in nicotine and/or nornicotine biosynthesis can be generated using the teachings herein.

Additionally, the interfering RNAs described herein can comprise a plurality nucleotide sequences that are each complementary to different portions of the sense strand of a gene involved in nicotine and/or nornicotine biosynthesis. Alternatively, the interfering RNAs described herein can comprise a plurality nucleotide sequences that are each complementary to at least a portion of the sense strands of different genes involved in nicotine biosynthesis. Still further, a single RNAi construct or inhibition cassette can be used to inhibit a plurality of genes involved in the regulation of the production of nicotine and/or nornicotine. For example, as described below, it was found that the A622 inhibitory fragment and inhibition cassette (SEQ. ID. Nos. 2 and 3) efficiently reduced production of nicotine and nornicotine in some lines of tobacco and in other lines of tobacco conventional levels of nicotine were maintained but the amount of nornicotine in said tobacco was 0.00 mg/g. Still further, the PMTase inhibitory sequence and PMTase inhibition cassette (SEQ. ID. Nos. 4 and 5) were designed to complement common regions of a plurality of PMTase genes so that the production of multiple gene products can be inhibited or reduced with a single construct.

In preferred embodiments, the interfering RNAs described herein comprise at least one region of double-stranded RNA (duplex RNA). This duplex RNA can range from about 10 bp in length to about 10,000 bp in length. In some embodiments, the duplex RNA ranges from about 15 bp in length to about 1500 bp in length. In other embodiments, the duplex RNA ranges from about 20 bp in length to about 1200 bp in length. In still other embodiments, the duplex RNA ranges from about 21 bp in length to about 23 bp in length. In a preferred embodiment, the duplex RNA has a length of 22 bps. Short regions of duplex RNA are often designated siRNA, whereas longer regions of RNA duplex are often termed dsRNA. In some embodiments, the interfering RNA duplex region is a dsRNA. In other embodiments, the interfering RNA duplex region is an siRNA. In a preferred embodiment, the duplex region about the length of the coding sequence of a target mRNA encoding a polypeptide involved in nicotine biosynthesis.

Interfering RNAs described herein can be generated using a variety of techniques. For example, an interfering RNA can be generated in a host cell in vivo by providing the cell with one or more a nucleic acid constructs that comprise the nucleic acids necessary to encode the strands of a double-stranded RNA. Such constructs can be included in various types of vectors. Exemplary vectors contemplated herein include, but are not limited to, plasmids, viral vectors, viroids, replicable and nonreplicable linear DNA molecules, replicable and nonreplicable linear RNA molecules, replicable and nonreplicable circular DNA molecules and replicable and nonreplicable circular RNA molecules. Preferred vectors include plasmid vectors, especially vector systems derived from the Agrobacterium Ti plasmid, such as pCambia vectors and derivatives thereof.

In some embodiments, both strands of the double-stranded region of the interfering RNA can be encoded by a single vector. In such cases, the vector comprises a first promoter operably linked to a first nucleic acid which is substantially similar or identical to at least a portion of the target mRNA. The vector also comprises a second promoter operably linked to a second nucleic acid, which is complementary or substantially to the first nucleic acid.

Another type of single vector construct, which can be used to generate interfering RNA, encodes a double-stranded RNA hairpin. In such embodiments, the vector comprises a promoter operably linked to a nucleic acid that encodes both strands of the duplex RNA. The first nucleotide sequence, which encodes the strand that is substantially similar or identical to at least a portion of the target mRNA, is separated from the second nucleotide sequence, which encodes a strand complementary or substantially complementary to the first strand, by a region of nucleotide sequence that does not substantially hybridize with either of the strands. This nonhybridizing region permits the RNA sequence transcribed from the vector promoter to fold back on itself, thereby permitting the complementary RNA sequences to hybridize so as to produce an RNA hairpin. Vectors comprising a plurality of nucleic acids, each of which encode both strands of the duplex RNA are also contemplated.

Other embodiments relate to multiple vector systems for the production of interfering RNA. In one example, a multiple vector system is used to produce a single interfering RNA that is specific for a single gene product involved in nicotine biosynthesis. In such embodiments, at least two vectors are used. The first vector comprises a promoter operably linked to a first nucleic acid that encodes a first strand of the RNA duplex that is present in the interfering RNA. The second vector comprises a promoter operably linked to a second nucleic acid that encodes the second strand of the RNA duplex, which is complementary to the first strand.

Other multiple vector systems are combinations of vectors, wherein each vector in the system encodes a different interfering RNA. Each of the interfering RNAs is specific for different gene products involved in nicotine biosynthesis. In some embodiments, the vectors in a multiple vector system can encode different interfering RNAs that are specific to different portions of a single gene product involved in nicotine biosynthesis.

It will be appreciated that the promoters used in the above-described vectors can either be constitutive or regulated. Constitutive promoters are promoters that are always expressed. The constitutive promoters selected for use in the above-described vectors can range from weak promoters to strong promoters depending on the desired amount of interfering RNA to be produced. Regulated promoters are promoters for which the desired level of expression can be controlled. An example of a regulated promoter is an inducible promoter. Using an inducible promoter in the above-described vector constructs permits expression of a wide range of concentrations of interfering RNA inside a cell.

It will also be appreciated that there is no requirement that the same or same types of promoters be used in vectors or multiple vector systems that comprise a plurality of promoters. For example, in some vectors or vector systems, a first promoter, which controls the expression of the first interfering RNA strand, can be an inducible promoter, whereas the second promoter, which controls the expression of the second RNA strand, can be a constitutive promoter. This same principal can also be illustrated in a multiple vector system. For example, a multiple vector system may have three vectors each of which includes one or more different types of promoters. Such a system can include, for example, a first vector having repressible promoter that controls the expression of an interfering RNA specific for a first gene product involved in nicotine biosynthesis, a second vector having a constitutive promoter that controls the expression of an interfering RNA specific for a second gene product involved in nicotine biosynthesis and a third vector having an inducible promoter that controls the expression of an interfering RNA specific for a third gene product involved in nicotine biosynthesis.

In other embodiments, interfering RNAs can be produced synthetically and introduced into a cell by methods known in the art. Synthetic interfering RNAs can include a variety of RNA molecules, which include, but are not limited to, nucleic acids having at least one region of duplex RNA. The duplex RNA in such molecules can comprise, for example, two antiparallel RNA strands that form a double-stranded RNA having flush ends, two antiparallel RNA strands that form a double-stranded RNA having at least one end that forms a hair pin structure, or two antiparallel RNA strands that form a double-stranded RNA, wherein both ends form a hair pin structure. In some embodiments, synthetic interfering RNAs comprise a plurality of RNA duplexes.

The regions of RNA duplex in synthetic interfering RNAs can range from about 10 bp in length to about 10,000 bp in length. In some embodiments, the duplex RNA ranges from about 15 bp in length to about 1500 bp in length. In other embodiments, the duplex RNA ranges from about 20 bp in length to about 1200 bp in length. In still other embodiments, the duplex RNA ranges from about 21 bp in length to about 23 bp in length. In a preferred embodiment, the duplex RNA has a length of 22 bps. In preferred embodiments, synthetic interfering RNAs are siRNAs. In another preferred embodiment, the synthetic interfering RNA is an siRNA specific for the coding sequence of a target mRNA encoding a polypeptide involved in nicotine biosynthesis.

Some aspects of the present invention relate to interfering nucleic acids that are not comprised entirely of RNA. Still other aspects relate to interfering nucleic acids that do not comprise any RNA. Such interfering nucleic acids are synthetic interfering RNA analogs. These analogs substantially mimic the specificity and activity of interfering RNA from which they are modeled; however, they typically include additional properties which make their use desirable. For example, one or both strands of the interfering nucleic acid may contain one or more nonnatural nucleotide bases that improve the stability of the molecule, enhance that affinity of the molecule for the target mRNA and/or enhance cellular uptake of the molecule. Other modifications are also contemplated. For example, an interfering nucleic acid can include one or more nucleic acid strands composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as non-naturally-occurring nucleobases, sugars and covalent internucleoside linkages.

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure. Within the nucleic acid structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of interfering nucleic acids useful in certain embodiments can include one or more nucleic acid strands containing modified backbones or non-natural internucleoside linkages. As used herein, nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

In some embodiments, modified nucleic acid backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Certain nucleic acids having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

In some embodiments, modified nucleic acid backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

In other embodiments, the interfering nucleic acid can comprise one or more mimetic regions, wherein both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. In such embodiments, the base units are maintained for hybridization with an appropriate nucleic acid target compound. One such compound, a mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331: and 5,719,262, each of which is herein expressly incorporated by reference in its entirety. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

In still other embodiments, interfering nucleic acids may include nucleic acid strands having phosphorothioate backbones and/or heteroatom backbones. Modified interfering nucleic acids may also contain one or more substituted sugar moieties. In some embodiments, the interfering nucleic acids comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂ and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Another modification includes 2′-methoxyethoxy (2′ OCH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504).

Embodiments also include Locked Nucleic Acids (LNAs), which generate interfering nucleic acids having enhanced affinity and specificity for the target polynucleotide. LNAs are nucleic acid in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)n group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226, the disclosures of which are incorporated herein by reference in their entireties.

Other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Interfering nucleic acids may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

The interfering nucleic acids contemplated herein may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine. 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrimido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993, the disclosures of which are incorporated herein by reference in their entireties. Certain of these nucleobases are particularly useful for increasing the binding affinity of the interfering nucleic acids described herein. These include 5-substituted pyrimidines. 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Another modification of the interfering nucleic acids described herein involves chemically linking to at least one of the nucleic acid strands one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the of the interfering nucleic acid. The interfering nucleic acids can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include intercalators, reporter molecules, polyamines, polyamides. polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of nucleic acids, and groups that enhance the pharmacokinetic properties of such molecules. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve interfering nucleic acid uptake, enhance its resistance to degradation, and/or strengthen sequence-specific hybridization with target molecules. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve the uptake, distribution, metabolism or excretion of the interfering nucleic acid. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277, 923-937).

As described above, it is not necessary for all positions in a given compound to be uniformly modified, and in fact, more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an nucleic acid. The methods described herein also contemplate the use of interfering nucleic acids which are chimeric compounds. “Chimeric” interfering nucleic acid compounds or “chimeras,” as used herein, are interfering nucleic acid compounds, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid compound. These interfering nucleic acids typically contain at least one region wherein the nucleic acid is modified so as to confer upon the interfering nucleic acid increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleic acid may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase II is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby contributes further to the inhibition of gene expression by the interfering nucleic acid.

The above-described interfering nucleic acids may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare nucleic acids such as the phosphorothioates and alkylated derivatives. The interfering nucleic acid compounds for use with the methods described herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound. Although terms, such as interfering RNA, dsRNA and siRNA, are used throughout the remainder of the specification, it will be appreciated that in the context of synthetically produced interfering nucleic acids, that such terms are meant to include interfering nucleic acids of all types, including those which incorporate modifications, such as those described above.

The reduction in or elimination of the expression of genes and/or gene products involved in nicotine and/or nornicotine biosynthesis can be characterized by comparing the amount of nicotine and/or nornicotine produced in genetically modified cells, with the amount of nicotine and/or nornicotine produced in cells that have not been genetically modified. Alternatively, such reduction in or elimination of gene expression can be characterized by genetically analyzing plant cells so as to determine the level of mRNA present in the genetically modified plant cell as compared to a non-modified plant cell. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of reduction in gene expression, which can be greater than 10%, 25%, 33%, 50%, 90%, 95% or 99% as compared to an untreated cell. As with nicotine and nornicotine, the reduction in or elimination of TSNA production in tobacco can be characterized by comparing the amount of TSNAs produced genetically modified cells, with the amount of TSNAs produced in cells that have not been genetically modified. The section below provides more description of the transgenic plants and cells of the invention.

Transgenic Plant Cells and Plants

DNA sequences provided herein can be transformed into a variety of host cells. A variety of suitable host cells, having desirable growth and handling properties, are readily available in the art. As used herein, the term “gene” refers to a DNA sequence that incorporates (1) upstream (5′) regulatory signals including the promoter, (2) a coding region specifying the product, protein or RNA of the gene, (3) downstream regions including transcription termination and polyadenylation signals and (4) associated sequences required for efficient and specific expression. The DNA sequence of the present invention may consist essentially of the sequence provided herein, or equivalent nucleotide sequences representing alleles or polymorphic variants of these genes, or coding regions thereof. Use of the phrase “substantial sequence similarity” or “substantially similar” in the present specification and claims means that DNA, RNA or amino acid sequences which have slight and non-consequential sequence variations from the actual sequences disclosed and claimed herein are considered to be equivalent to the sequences of the present invention. In this regard, “slight and non-consequential sequence variations” mean that “similar” sequences (i.e., the sequences that have substantial sequence similarity with the DNA, RNA or proteins disclosed and claimed herein) will be functionally equivalent to the sequences disclosed and claimed in the present invention. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein.

As used herein, a “native nucleotide sequence” or “natural nucleotide sequence” means a nucleotide sequence that can be isolated from non-transgenic cells or tissue. Native nucleotide sequences are those which have not been artificially altered, such as by site-directed mutagenesis. Once native nucleotide sequences are identified, nucleic acid molecules having native nucleotide sequences may be chemically synthesized or produced using recombinant nucleic acid procedures as are known in the art. As used herein, a “native plant nucleotide sequence” is that which can be isolated from non-transgenic plant cells or tissue. As used herein, a “native tobacco nucleotide sequence” is that which can be isolated from non-transgenic tobacco cells or tissue. Use of the phrase “isolated” or “substantially pure” in the present specification and claims as a modifier of nucleic acids, polypeptides or proteins means that the nucleic acids, polypeptides or proteins so designated have been separated from their in vivo cellular environments through the efforts of human beings.

DNA constructs, or “transcription cassettes,” of the present invention may include, 5′ to 3′ in the direction of transcription, a promoter as discussed herein, a DNA sequence as discussed herein operatively associated with the promoter, and, optionally, a termination sequence including stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.

The term “operatively associated,” as used herein, refers to nucleotide sequences on a single nucleic acid molecule which are associated so that the function of one is affected by the other. Thus, a promoter is operatively associated with a nucleotide sequence when it is capable of affecting the transcription of that sequence (i.e., the nucleotide sequence is under the transcriptional control of the promoter). The promoter is said to be “upstream” from the transcribed nucleotide sequence, which is in turn said to be “downstream” from the promoter.

In embodiments, wherein a termination signal is used, any suitable termination signal may be employed in carrying out the present invention, examples thereof including, but not limited to, the nopaline synthase (nos) terminator, the octopine synthase (ocs) terminator, the CaMV terminator, or native termination signals derived from the same gene as the transcriptional initiation region or derived from a different gene. See, e.g., Rezian et al. (1988) supra, and Rodermel et al. (1988), supra. Alternatively, if nicotine levels are decreased by molecular decoy technology rather than by antisense or other methods, the molecular decoy fragments, with or without additional sequences, may be provided to the plant cell by any means. For example, the molecular decoy fragment may have an accompanying gene encoding a selectable marker, other suitable genes, or may be present as part of a plasmid vector. The molecular decoy fragment may consist of a single or double stranded DNA or RNA molecule. The molecular decoy may be integrated into the genome or may exist freely in the cell.

The transcription cassette may be provided in a DNA construct that also has at least one replication system. For convenience, it is common to have a replication system functional in Escherichia coli, such as ColE1, pSC101, pACYC184, or the like. In this manner, at each stage after each manipulation, the resulting construct may be cloned, sequenced, and the correctness of the manipulation determined. In addition, or in place of the E. coli replication system, a broad host range replication system may be employed, such as the replication systems of the P-1 incompatibility plasmids, e.g., pRK290. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be employed for selection in a prokaryotic host, while another marker may be employed for selection in a eukaryotic host, particularly the plant host. The markers may be protection against a biocide, such as antibiotics, toxins, heavy metals, or the like; may provide complementation, by imparting prototrophy to an auxotrophic host; or may provide a visible phenotype through the production of a novel compound in the plant.

The various fragments comprising the various constructs, transcription cassettes, markers, and the like may be introduced consecutively by restriction enzyme cleavage of an appropriate replication system, and insertion of the particular construct or fragment into the available site. After ligation and cloning the DNA construct may be isolated for further manipulation. All of these techniques are amply exemplified in the literature as exemplified by J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).

Vectors which may be used to transform plant tissue with nucleic acid constructs of the present invention include both Agrobacterium vectors and ballistic vectors, as well as vectors suitable for DNA.-mediated transformation. The term ‘promoter’ refers to a region of a DNA sequence that incorporates the necessary signals for the efficient expression of a coding sequence. This may include sequences to which an RNA polymerase binds but is not limited to such sequences and may include regions to which other regulatory proteins bind together with regions involved in the control of protein translation and may include coding sequences.

The recombinant DNA molecules and vectors used to produce the transformed tobacco cells and plants may further comprise a dominant selectable marker gene. Suitable dominant selectable markers for use in tobacco include, inter alia, antibiotic resistance genes encoding neomycin phosphotransferase (NPTII), and hygromycin phosphotransferase (HPT). Other well-known selectable markers that are suitable for use in tobacco include a mutant dihydrofolate reductase gene that encodes methotrexate-resistant dihydrofolate reductase. DNA vectors containing suitable antibiotic resistance genes, and the corresponding antibiotics, are commercially available.

Aspects of the present invention concern transgenic plant cells comprising one or more interfering RNAs that are capable of reducing or eliminating the expression of one or more target genes and/or target gene products involved in nicotine and/or nornicotine, biosynthesis. As described above, an appropriate interfering RNA comprises a duplex RNA that comprises a first strand that is substantially similar or identical to at least a portion of a target gene or target mRNA, which encodes a gene product involved in nicotine, and/or nornicotine biosynthesis. The RNA duplex also comprises a second strand that is complementary or substantially complementary to the first strand. Examples 6-18 describe the use of several preferred RNAi constructs.

The interfering RNA or nucleic acid construct comprising the interfering RNA can be introduced into the plant cell in any suitable manner. Plant cells possessing stable interfering RNA activity, for example, by having a nucleic acid construct stably integrated into a chromosome, can be used to regenerate whole plants using methods known in the art. As such, some aspects of the present invention relate to plants, such as tobacco plants, transformed with one or more nucleic acid constructs and/or vectors which encode at least one interfering RNA that is capable of reducing or eliminating the expression of a gene product involved in nicotine biosynthesis. Transgenic tobacco cells and the plants described herein are characterized in that they have a reduced amount of nicotine, nornicotine, and/or TSNA and/or generate a reduced amount of PAHs upon pyrolysis, as compared to unmodified or control tobacco cells and plants.

The tobacco plants described herein are suitable for conventional growing and harvesting techniques (e.g. topping or no topping, bagging the flowers or not bagging the flowers, cultivation in manure rich soil or without manure) and the harvested leaves and stems are suitable for use in any traditional tobacco product including, but not limited to, pipe, cigar and cigarette tobacco and chewing tobacco in any form including leaf tobacco, shredded tobacco or cut tobacco. It is also contemplated that the low nicotine and/or TSNA tobacco described herein can be processed and blended with conventional tobacco so as to create a wide-range of tobacco products with varying amounts of nicotine and/or nitrosamines.

Gene silencing has been employed in several laboratories to create transgenic plants characterized by lower than normal amounts of specific gene products. As used herein, “exogenous” or “heterologous” nucleic acids, including DNAs and/or RNAs, refer to nucleic acids that have been introduced into a cell (or the cell's ancestor) through the efforts of humans. The nucleic acid constructs that are used with the transgenic plants and the methods for producing the transgenic plants described herein encode one or more interfering RNA constructs comprising regulatory sequences, which include, but are not limited to, a transcription initiation sequence (“promoter”) operable in the plant being transformed, and a polyadenylation/transcription termination sequence. Typically, the promoter is located upstream of the 5′-end of the nucleotide sequence to be expressed. The transcription termination sequence is generally located just downstream of the 3′-end of the nucleotide sequence to be transcribed.

In some preferred embodiments, the nucleic acid encoding the exogenous interfering RNA, which is transformed into a tobacco cell, comprises a first RNA strand that is identical to the an endogenous coding sequence of a gene encoding a gene product involved in nicotine biosynthesis. However, minor variations between the exogenous and endogenous sequences can be tolerated. It is preferred, but not necessarily required, that the exogenously-produced interfering RNA sequence, which is substantially similar to the endogenous gene coding sequence, be of sufficient similarity to the endogenous gene coding sequence, such that the complementary interfering RNA strand is capable of binding to the endogenous sequence in the cell to be regulated under stringent conditions as described below.

In some embodiments, the heterologous sequence utilized in the methods described herein may be selected so as to produce an interfering RNA product comprising a first strand that is substantially similar or identical to the entire mRNA sequence of a gene involved in nicotine biosynthesis, or to a portion thereof, and a second strand that is complementary to the mRNA sequence, or to a portion thereof. The interfering RNA may be complementary to any contiguous sequence of the natural messenger RNA. For example, it may be complementary to the endogenous mRNA sequence proximal to the 5′-terminus or capping site, downstream from the capping site, between the capping site and the initiation codon and may cover all or only a portion of the non-coding region, may bridge the non-coding and coding region, be complementary to all or part of the coding region, complementary to the C-terminus of the coding region or complementary to the 3′-untranslated region of the mRNA.

The nucleotide sequences provided herein, such as interfering RNAs or nucleic acids encoding interfering RNAs, can be transformed into a variety of host cells. As used herein, “transformation” refers to the introduction of exogenous nucleic acid into cells so as to produce transgenic cells stably transformed with the exogenous nucleic acid. A variety of suitable host cells, having desirable growth and handling properties, are readily available in the art.

Standard techniques, such as restriction mapping, Southern blot hybridization, polymerase chain reaction (PCR) and/or nucleotide sequence analysis can be employed to identify clones expressing the desired interfering RNA construct. Following the introduction and verification of the desired interfering RNA or nucleic acid construct encoding the desired interfering RNA, whole plants can be regenerated from successfully transformed cells using conventional techniques.

“Transcription cassettes” encoding the interfering RNAs that are used to produce the transgenic cells and plants of the present invention include, 5′ to 3′ in the direction of transcription, a promoter as described herein, a nucleotide sequence as described herein operatively associated with the promoter, and, optionally, a termination sequence including stop signal for RNA polymerase and a polyadenylation signal. All of these regulatory regions should be capable of operating in the cells of the tissue to be transformed. Any suitable termination signal may be employed in carrying out the present invention, examples thereof including, but not limited to, the nopaline synthase (nos) terminator, the octapine synthase (ocs) terminator, the CaMV terminator or native termination signals, derived from the same gene as the transcriptional initiation region or derived from a different gene. (See, e.g., Rezian et al. (1988) supra, and Rodermel et al. (1988), supra).

Promoters employed in carrying out the invention may be constitutively active promoters. Numerous constitutively active promoters that are operable in plants are available. A preferred example is the Cauliflower Mosaic Virus (CaMV) 35S promoter, which is expressed constitutively in most plant tissues. As an alternative, the promoter may be a root-specific promoter or root cortex specific promoter, as explained in greater detail below.

Nucleic acid sequences have been expressed in transgenic tobacco plants utilizing the Cauliflower Mosaic Virus (CaMV) 35S promoter. (See, e.g., Cornelissen et al., “Both RNA Level and Translation Efficiency are Reduced by Anti-Sense RNA in Transgenic Tobacco”, Nucleic Acids Res. 17, pp. 833-43 (1989); Rezaian et al., “Anti-Sense RNAs of Cucumber Mosaic Virus in Transgenic Plants Assessed for Control of the Virus”, Plant Molecular Biology 11, pp. 463-71 (1988); Rodermel et al., “Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxylase Enzyme Levels in Transformed Tobacco Plants”, Cell 55, pp. 673-81 (1988); Smith et al., “Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes”, Nature 334, pp. 724-26 (1988); Van der Krol et al., “An Anti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation”, Nature 333, pp. 866-69 (1988)).

Use of the CaMV 35S promoter for expression of interfering RNAs in the transformed tobacco cells and plants of this invention is preferred. Use of the CaMV promoter for expression of other recombinant genes in tobacco roots has been well described (Lam et al., “Site-Specific Mutations Alter In Vitro Factor Binding and Change Promoter Expression Pattern in Transgenic Plants”, Proc. Nat. Acad Sci. USA 86, pp. 7890-94 (1989); Poulsen et al. “Dissection of 5′ Upstream Sequences for Selective Expression of the Nicotiana plumbaginifolia rbcS-8B Gene”, Mol. Gen. Genet. 214, pp. 16-23 (1988)). Other promoters that are active only in root tissues (root specific promoters) are also particularly suited to the methods of the present invention. See, e.g., U.S. Pat. No. 5,459,252 to Conkling et al.; Yamamoto et al., The Plant Cell, 3:371 (1991). The TobRD2 root-cortex specific promoter may also be utilized. All patents cited herein are intended to be incorporated herein by reference in their entirety.

The recombinant interfering nucleic acid molecules and vectors used to produce the transformed tobacco cells and plants of this invention may further comprise a dominant selectable marker gene. Suitable dominant selectable markers for use in tobacco include, inter alia, antibiotic resistance genes encoding neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPT). Preferred selectable markers include the norflurazone resistance genes described in this disclosure. Other well-known selectable markers that are suitable for use in tobacco include a mutant dihydrofolate reductase gene that encodes methotrexate-resistant dihydrofolate reductase. DNA vectors containing suitable antibiotic resistance genes, and the corresponding antibiotics, are commercially available.

Transformed tobacco cells are selected out of the surrounding population of non-transformed cells by placing the mixed population of cells into a culture medium containing an appropriate concentration of the antibiotic (or other compound normally toxic to tobacco cells) against which the chosen dominant selectable marker gene product confers resistance. Thus, only those tobacco cells that have been transformed will survive and multiply. Additionally, the positive selection techniques described by Jefferson (e.g., WO 00055333; WO 09913085; U.S. Pat. Nos. 5,599,670; 5,432,081; and 5,268,463, hereby expressly incorporated by reference in their entireties) can be used.

Microparticles suitable for the ballistic transformation of a plant cell, carrying a nucleic acid construct of the present invention, are also useful for making the transformed plants described herein. The microparticle is propelled into a plant cell to produce a transformed plant cell and a plant is regenerated from the transformed plant cell. Any suitable ballistic cell transformation methodology and apparatus can be used in practicing the present invention. Exemplary apparatus and procedures are disclosed in Sanford and Wolf, U.S. Pat. No. 4,945,050, and in Christou et al., U.S. Pat. No. 5,015,580. When using ballistic transformation procedures, the transcription cassette may be incorporated into a plasmid capable of replicating in or integrating into the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The nucleic acid construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Plant species may be transformed with the interfering RNA,nucleic acid construct encoding an interfering RNA, or other DNA constructs of the present invention by the nucleic acid-mediated transformation of plant cell protoplasts. Plants may be subsequently regenerated from the transformed protoplasts in accordance with procedures well known in the art. Fusion of tobacco protoplasts with nucleic acid-containing liposomes or with nucleic acid constructs via electroporation is known in the art. (Shillito et al., “Direct Gene Transfer to Protoplasts of Dicotyledonous and Monocotyledonous Plants by a Number of Methods, Including Electroporation”, Methods in Enzymology 153, pp. 313-36 (1987)).

These inhibition constructs or RNAi constructs can be transferred to plant cells by any known method in the art. Preferably, Agrobacterium-mediated or Biolistic-mediated transformation are used, according to well-established protocols. It is also contemplated that Transbacter-mediated transformation can be used, as described below. (See Broothaerts et al., Nature 433, 629 (2005), herein expressly incorporated by reference in its entirety).

Methods of making recombinant plants of the present invention, in general, involve first providing a plant cell capable of regeneration (the plant cell typically residing in a tissue capable of regeneration). The plant cell is then transformed with a DNA construct comprising a transcription cassette of the present invention (as described herein) and a recombinant plant is regenerated from the transformed plant cell. As explained below, the transforming step is carried out by techniques as are known in the art, including but not limited to bombarding the plant cell with microparticles, carrying the transcription cassette, infecting the cell with an Agrobacterium tumefaciens containing a Ti plasmid carrying the transcription cassette or any other technique suitable for the production of a transgenic plant.

Numerous Agrobacterium vector systems useful in carrying out the present invention are known. For example, U.S. Pat. No. 4,459,355, herein expressly incorporated by reference, discloses a method for transforming susceptible plants, including dicots, with an Agrobacterium strain containing the Ti plasmid. The transformation of woody plants with an Agrobacterium vector is disclosed in U.S. Pat. No. 4,795,855, herein expressly incorporated by reference. Further, U.S. Pat. No. 4,940,838 to Schilperoort et al., herein expressly incorporated by reference, discloses a binary Agrobacterium vector (i.e., one in which the Agrobacterium contains one plasmid having the vir region of a Ti plasmid but no T region, and a second plasmid having a T region but no vir region) useful in carrying out the present invention.

As used herein, transformation refers to the introduction of exogenous DNA into cells, so as to produce transgenic cells stably transformed with the exogenous DNA. Transformed cells are induced to regenerate intact tobacco plants through application of tobacco cell and tissue culture techniques that are well known in the art. The method of plant regeneration is chosen so as to be compatible with the method of transformation. The stable presence and the orientation of the desired sequence in transgenic tobacco plants can be verified by Mendelian inheritance of the desired sequence, as revealed by standard methods of DNA analysis applied to progeny resulting from controlled crosses. After regeneration of transgenic tobacco plants from transformed cells, the introduced DNA sequence is readily transferred to other tobacco varieties through conventional plant breeding practices and without undue experimentation.

By this approach, first bacteria are prepared as follows. YM plus antibiotic plates (see below) are streaked with bacteria and the plates are incubated for 2-3 days at 28° C. Transformation is accomplished by measuring about 20 mL Minimal A medium for each bacterial strain. Scrapping or washing the Scrape or wash bacteria from plate with sterile loop and then suspending said bacteria in 20 mL of Minimal A medium. The cell density is adjusted to an OD600 0.9-1.0.

Next, the first healthy fully expanded leaves from 4-5 week old tissue culture grown tobacco plants are cut into 0.5cm squares (or can use a cork borer, which is about 1.0 cm diameter) in deep petri dish, under sterile RMOP liquid medium. The tissue pieces are stored in RMOP in a deep petri dish. The leaf pieces (about 20 per transformation) are then transferred to a deep petri dish containing bacterial suspension. To ensure that the bacteria have contacted a cut edge of the leaf, the suspension with leaf cutting is swirled and is left standing for 5 minutes. The leaf pieces are then removed from the suspension and blotted dry on filter paper or on the edge of the container. The leaf pieces are then placed with adaxial side (upper leaf surface) on solid RMOP at about 10 pieces per plate. The plates are then incubated in the dark at 28° C. for: 2-3 days, if A. tumefaciens is used, 5 days if S. meliloti is used, 5 days M. loti is used, and 5-11 days if Rhizobium sp. NGR234 is used.

Over the next week, selection is performed. For the purposes of this example, hygromycin selction is performed. Accordingly, the leaf pieces are transferred onto solid RMOP-TCH, with abaxial surface (lower surface of leaf) in contact with media. The plates are incubated for 2-3 weeks in the light at 28° C., with 16 hours daylight per day. Subculture occurs every 2 weeks.

Plantlet formation is accomplished as follows. Once shoots appear, the plantlet is transferred to MST-TCH pots. The plantlets are grown with 16 hours daylight for 1-2 weeks. Once roots form the plants appear, the plants can be transferred to soil in the greenhouse.

Media and Solutions for Tobacco Transformation. YM Media (1 L) Mannitol 10 g Yeast extract 0.4 g K2HPO4 (10% w/v stock) 1 ml KH2PO4 (10% w/v stock) 4 ml NaCl (10% w/v stock) 1 ml MgSO4.7H2O (10% w/v stock) 2 ml pH 6.8 Agar 15 g/L Autoclave

*When ready to pour add antibiotic selection if required

Keep poured plates for 2 days at room temperature to visualize any contamination, then store at 4° C.

RMOP+RMOP-TCH Media

(Svab, Z., et al., 1975. Transgenic tobacco plants by cocultivation of leaf disks with pPZP Agrobacterium binary vectors. In “Methods in Plant Molecular Biology—A Laboratory Manual”, P. Maliga, D. Klessig, A. Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press: 55-77), herein expressly incorporated by reference in its entirety).

1L Final Conc. Sucrose 30 g (3%) Myo-inositol 100 mg (0.1%) MS Macro 10x 100 mL (1x) MS Micro 1000x 1 mL (1x) Fe2EDTA Iron 100x 10 mL (1x) Thiamine-HCl (10 mg/mL stock) 100 μL (1 mg) NAA (1 mg/mL stock) 100 μL 0.1 mg) BAP (1 mg/mL stock) 1 mL (1 mg) pH 5.8 Phytagel 2.5 g/L for solid autoclave

*for RMOP-TCH, when ready to pour add:

Timentin (200 mg/mL stock) 1 mL, Claforan (250 mg/mL stock) 1 mL, and Hygromycin (50 mg/mL stock) 1 mL

BAP (1 mg/ml) (6-Benzylaminopurine)

Add 1N KOH drop wise to 100 mg BAP until dissolved. Make up to 100 mL with Milli-Q H2O and store at 4° C.

NAA (1 mg/ml) (Naphthalene Acetic Acid)

Dissolve 100 mg NAA in 1 mL absolute ethanol. Add 3 mL 1N KOH. Make up to 80 mL with Milli-Q H2O. Adjust pH to 6.0 with 1N HCl, make up to 100 mL with Milli-Q H2O, and store at 4° C.

Cefotaxamine (250 mg/ml)

Add 8 ml sterile Milli-Q H2O to 2 g Claforan and store at 4° C. in dark

Timentin (200 mg/ml)

Add 15 ml sterile Milli-Q H2O to 3 g Timentin and store at 4° C.

MST+MST-TCH Media

(Svab, Z., et al., 1975. Transgenic tobacco plants by cocultivation of leaf disks with pPZP Agrobacterium binary vectors. In “Methods in Plant Molecular Biology—A Laboratory Manual”, P. Maliga, D. Klessig, A., Cashmore, W. Gruissem and J. Varner, eds. Cold Spring Harbor Press: 55-77), herein expressly incorporated by reference in its entirety).

1L Final Concentration Sucrose 30 g (3%) MS Macro 10x 100 mL (1x) MS Micro 1000x 1 mL (1x) Fe2EDTA Iron 100x 10 mL (1x) pH 5.8 Phytagel 2.5 g/L Autoclave For MST-TCH, when ready to pour add: Timentin (200 mg/mL stock) (1 mL) Cefotaxamine (250 mg/mL stock) (1 mL) Hygromycin (50 mg/mL stock) (1 mL)

MS Macro 10× ((Murashige and Skoog., Phys. Plant. 15: 473-497 (1962), herein expressly incorporated by reference in its entirety)).

Final Concentration 10x (g/L) KNO3 19.0 NH4 N03 16.5 CaCl2.2H2O 4.4 MgS04.7H2O 3.7 KH2PO4 1.7 Store 4° C.

Substituting Chemicals.

CaCl2 3.3 g/L

MgSO4 1.8 g/L

MS Micro 1000×

(Murashige and Skoog., Phys. Plant. 15: 473-497 (1962), herein expressly incorporated by reference in its entirety).

Final Concentration 1000x (g/L) MnS04.4H20 22.3 ZnS04.7H20 8.6 H3BO3 6.2 KI 0.83 Na2MoO4.2H2O 0.25 CuSO4.5H2O 25 mg CoCl2.6H2O 25 mg Store 4° C.

Substituting Chemicals:

MnS04.H20 16.9/L

FeSO4EDTA Iron 100× (g/1 L) FeS04.7H20 2.78 Na2EDTA 3.72 Store 4° C. in dark bottle

Once the transformed cells are selected, by any of the approaches described above, they are induced to regenerate intact tobacco plants through application of tobacco cell and tissue culture techniques that are well known in the art. The method of plant regeneration is chosen so as to be compatible with the method of transformation. The stable presence of an interfering RNA or a nucleic acid encoding an interfering RNA in transgenic tobacco plants can be verified by Mendelian inheritance of the interfering RNA or a nucleic acid encoding an interfering RNA sequence, as revealed by standard methods of nucleic acid analysis applied to progeny resulting from controlled crosses. After regeneration of transgenic tobacco plants from transformed cells, the introduced nucleic acid sequence can be readily transferred to other tobacco varieties through conventional plant breeding practices and without undue experimentation.

For example, to analyze the segregation of the transgene, regenerated transformed plants (TO) may be grown to maturity, tested for nicotine and/or TSNA levels, and selfed to produce T₁ plants. A percentage of T₁ plants carrying the transgene are homozygous for the transgene. To identify homozygous T₁ plants, transgenic T₁ plants are grown to maturity and selfed. Homozygous T₁ plants will produce T₂ progeny where each progeny plant carries the transgene; progeny of heterozygous T₁, plants will segregate 3:1.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a nucleic acid embodiment of the present invention. Preferred plants for introduction of a nucleic acid embodiment, described herein, include Nicotiana. Preferred varieties of Nicotiana for introduction of a nucleic acid embodiment as described herein include the Nicotiana tabacum varieties provided in Table 1. TABLE 1 Burley Dark One Newest Varieties Varieties Flu Cured Other Virginia Hybrid Sucker Varieties Oriental KT 200 BLACK K 149 CU 748 BROWN NBH 98 OS400 GL 350 D174 LC MAMMOTH LEAF MS LIZARD KT 204 DF 485 K 326 GL 737 TAIL 21 × KY KY LC ORNOCO 10 160 LIZARD KY DF 911 K 346 OX 207 TAIL MS TURTLE 14 × KY FOOT L8 KY 10 DT 508 K 394 PVH 03 M and N TN 97 KY 14 DT 518 K 730 PVH 09 SHIREY KT 200 Coker 371 PVH WALKER KY 17 DT 592 Gold 2040 BROADLEAF KY 907 GREEN CU 748 RG 17 WOOD KY 907 IMPROVED GL 737 RG 81 LC MADOLE KY 908 KT-D4 LC GL 939 RGH 4 KY 908 KY 160 GL 973 RGH 51 KY 910 KY 171 K 358 RS 1410 MS KY 171 K 399 Speight Burley 21 × KY 10 168 MS KY14 × L8 LITTLE NC 102 Speight CRITTENDEN 179 N 126 LITTLE NC 291 Speight WOOD 190 NARROW N 777 LEAF NC 297 Speight MADOLE 196 N 88 NEWTON'S NC 55 Speight VH MADOLE 200A NBH 98 NL MADOLE NC 606 Speight 210 TN 86 TN D94 NC 71 Speight 218 TN 86 LC TN D950 NC 72 Speight 220 TN 90 TR MADOLE NC 810 Speight H-20 TN 90 LC VA 309 RGH 4 Speight H-6 TN 97 LC VA 312 RGH 51 Speight NF-3 VA 509 VA 355 VA 119 LA21 VA 359 NC 37 NF OX 414 NF Sp. G- 172

The term “organogenesis,” as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term “embryogenesis,” as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems) and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the transcription cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or T₁) transformed plants may be selfed to give homozygous second generation (or T₂) transformed plants and the T₂ plants further propagated through classical breeding techniques. A dominant selectable marker (such as nptII) can be associated with the transcription cassette to assist in breeding.

As used herein, a crop comprises a plurality of plants of the present invention, and of the same genus, planted together in an agricultural field. By “agricultural field” is meant a common plot of soil or a greenhouse. Thus, aspects of the present invention provide a method of producing a crop of plants having reduced amounts of nicotine and/or nornicotine, as compared to a similar crop of non-transformed plants of the same species and variety.

The modified tobacco plants described herein are suitable for conventional growing and harvesting techniques (e.g. topping or no topping, bagging the flowers or not bagging the flowers, cultivation in manure rich soil or without manure). The harvested tobacco leaves and stems are suitable for conventional methods of processing such as curing and blending. The modified tobacco is suitable for use in any traditional tobacco product including, but not limited to, pipe, cigar and cigarette tobacco, and chewing tobacco in any form including leaf tobacco, shredded tobacco, or cut tobacco.

Some embodiments concern the production and identification of particular lines of a transgenic Burley variety (Vector 21-41), which have very low levels of nicotine and TSNAs. The constructs used to create these particular lines of transgenic Burley tobacco are provided in Conkling et al., W098/56923; U.S. Pat. Nos.: 6,586,661; 6,423,520; and U.S. patent application Ser. Nos. 09/963,340; 10/356,076; 09/941,042; 10/363,069; 10/729,121; 10/943,346, all of which are hereby expressly incorporated by reference in their entireties. After the creation and analysis of nearly 2,000 lines of transgenic Burley tobacco, these particular lines of reduced nicotine and TSNA transgenic tobacco were identified. Tobacco harvested from these lines were incorporated into tobacco products (Quest 1®, Quest 2®, and Quest 3®) and were analyzed for their ability to reduce the potential to contribute to a tobacco-related disease, as described in the sections above. It was found that tobacco products comprising these lines of transgenic Burley tobacco, had a reduced potential to contribute to a tobacco-related disease (i.e., that these tobacco products are reduced risk tobacco products).

Several embodiments concern isolated nucleic acids that comprise, consist, or consist essentially of the nucleic acids described in the sequence listing (SEQ. ID. NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) and fragments thereof at least 30 consecutive nucleotides in length. That is, embodiments of the invention include an isolated nucleic acid comprising, consisting of, consisting essentially of, any one or more of the sequences of SEQ. ID. NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, or a fragment thereof (e.g., a fragment that is at least, less than or equal to or greater than 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, or 9000 consecutive nucleotides of SEQ. ID. NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

In preferred embodiments, the target gene or target mRNA encodes QPTase, PMTase, or the A622 gene product. In preferred embodiments, an interfering RNA comprises, consists, or consists essentially of an RNA strand that is complementary to at least a portion (e.g., less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, or 1000 consecutive nucleotides) of SEQ ID NOS: 1, 6, 4, or 2, and inhibits the production of QPTase, PMTase, A622, nicotine, nornicotine, NNN, NNK, NAT, or NAB in a tobacco. In related embodiments, the interfering RNA comprises, consists, or consists essentially of an RNA strand that is complementary to each least a portion (e.g., less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, or 1000 consecutive nucleotides) of SEQ ID NO: 2, and inhibits production of nornicotine but not nicotine in a tobacco.

Some of these nucleic acid embodiments comprise, consist, or consist essentially of fragments of the QPTase, PMTase, and A622 genes that were found to inhibit gene expression unexpectedly well in the RNAi constructs described herein, producing reduced alkaloid tobacco (below 7,000 ppm, 1,000 ppm, or 500 ppm).

Still more of the nucleic acid embodiments concern several phytoene desaturase (PDS) mutants (e.g., PDSM-1, PDSM-2, and PDSM-3, SEQ. lD. NOs.: 7, 8, or 9) that were developed to confer resistance to norflurazone, which allows both tissue-culture selection of cells transformed with the construct, as well as, field-based selection, wherein weeds and tobacco, which do not contain an herbicide resistance gene, are removed from the field or crop by spraying the herbicide norflurazone or an herbicide of the same class or activity (e.g., herbicides that contain C₁₂H₉ClF₃N₃O (see U.S. Pat. No. 3,644,355, herein expressly incorporated by reference in its entirety), but plants expressing PDSM-1, PDSM-2, or PDSM-3 survive the herbicide contact). That is, some embodiments include isolated nucleic acids that comprise, consist, or consist essentially of the PDS mutant sequences provided by SEQ. ID. NOs.:7, 8, or 9 and fragments thereof at least 30 nucleotides in length (e.g., less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1729 consecutive nucleotides) that include a mutation (e.g., T1478G, which encodes Val493Gly; G863C, which encodes Arg288Pro; and T1226C, which encodes Leu409Pro) that confers resistance to norflurazone). Preferably, the fragments of the PDS mutants described herein confer resistance to norflurazone, although fragments that do not confer resistance to the herbicide are also useful in the field in assays designed to follow the retention of constructs described herein in successive generations of transgenic plants. Approaches to develop more norflurazone-resistance genes are also provided herein.

Additional embodiments include isolated nucleic acids that comprise, consist, or consist essentially of root-specific promoters, constitutive promoters, and developmentally regulated promoters, which can be used interchangeably with the nucleic acid sequences described herein. Some embodiments, for example, include a root-specific promoter such as the truncated RD2 promoter (SEQ. ID NO. 10) or the Putrescene methyl transferase promoter (PMT-1) (SEQ. ID NO. 11). Constitutive promoters that can be used with embodiments described herein include the GapC promoter (SEQ. ID. NO.: 12), Actin 2 promoter (Act2P) (SEQ. ID NO. 13), the tobacco alcohol dehydrogenase promoter (ADP) (SEQ. ID NO. 14), and the Arabidopsis ribosomal protein L2 promoter (RPL2P) (SEQ. ID NO. 15). Developmentally regulated promoters that can be used with the nucleic acid sequences described herein include the cinnamyl alcohol dehydrogenase promoter (SEQ. ID NO. 16) and the metallothionein I promoter (SEQ. ID NO. 17). Additional embodiments also include isolated nucleic acids that comprise, consist, or consist essentially of the GAD2 terminator (SEQ. ID NO. 18), a FAD2 intron (provided by (SEQ. ID NO. 19), which was used as a spacer in several of the RNAi constructs, and the PAP1 intron (provided by nucleotides 6446-7625 of (SEQ. ID NO. 20). Because of the unique properties of the FAD2 intron, in particular the hair-pin secondary structure afforded by the interaction of splice sites in the sequence, it was found, unexpectedly, that transgenic tobacco could be made with various inhibitory sequences with nearly equivalent success (e.g., approximately 50% of the reduced nicotine lines created by multiple constructs were found to have less than 1,000 ppm total alkaloid). Accordingly, significantly improved RNAi constructs were generated using this spacer. That is, aspects of the invention concern the use of an intronic sequence comprising splicing recognition sequences (preferably FAD2 or PAP1 intron) to link or join a first RNA sequence to a second RNA sequence that is complementary to said first RNA sequence, wherein said first or second RNA sequence is complementary to a target RNA, which, preferably, regulates the production of a harmful compound in tobacco (e.g., nicotine or nornicotine).

Aspects of the invention also concern isolated nucleic acids that comprise, consist, or consist essentially of the inhibition and selection cassettes identified as SEQ. ID. Nos. 21, 22, 5, 3, 23, 24, or 25 and fragments thereof (e.g., a fragment that is at least, less than or equal to or greater than 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, or 9000 consecutive nucleotides) of SEQ. ID. Nos. 21, 22, 5, 3, 23, 24, or 25).

Aspects of the invention also concern isolated nucleic acids that comprise, consist, or consist essentially of a plurality of the nucleic acid sequences described herein. For example, a double knock-out construct comprising a portion of the A622 gene and a portion of the QPTase gene has been made and it is expected that this construct will efficiently reduce expression of at least two genes involved in the synthesis or regulation of the production of nicotine (SEQ. ID. No. 23). Accordingly, aspects of the invention concern an isolated nucleic acid construct that inhibits the expression of a plurality of genes that regulate the production of nicotine. In some aspects of these embodiments, said isolated nucleic acid construct inhibits the expression of at least two nicotine biosynthesis genes.

It should also be understood that aspects of the invention concern tobacco generated by crossing the transgenic tobaccos described herein. For example, some embodiments concern progeny of a cross between a transgenic tobacco having a reduced amount of nicotine created by different methods. Crossings of the transgenic tobacco described herein and wild-type tobacco are also aspects of the invention.

The interfering RNAs used with the embodied nucleic acids can be expressed from nucleic acid construct that encodes one or more strands of the RNA duplex of the interfering RNA. In some embodiments, the nucleic acid construct is present on a vector. The vectors may be viral vectors, plasmids, or any other vehicles for nucleic acid delivery. In other embodiments, the interfering RNAs described herein can be generated synthetically by methods, such as direct synthesis or in vitro transcription. In some embodiments, synthetic interfering nucleic acids comprising modified nucleic acids are contemplated. Other embodiments of the present invention include multiple vector systems for producing an interfering RNA wherein a first vector encodes the first strand of the interfering RNA and a second vector encodes the second strand of the interfering RNA.

Still other embodiments relate to tobacco cells comprising one or more of the nucleic acid constructs described herein, which encode an interfering RNA that is specific for a gene product involved in nicotine biosynthesis. In such embodiments, the interfering RNA reduces or eliminates the expression of such gene product. Additional embodiments relate to tobacco cells comprising one or more interfering RNAs that are specific for a gene product involved in nicotine biosynthesis. In certain embodiments, the interfering RNAs are synthetic interfering RNAs.

Some embodiments relate to tobacco plants and cured tobacco products having a reduced amount or nicotine, nornicotine, and/or TSNAs. In such embodiments, reduction in nicotine, nornicotine, and/or TSNA amounts in the tobacco plants and cured tobacco products is mediated by an interfering RNA comprising an RNA duplex wherein at least 30 consecutive nucleotides (e.g., at least or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800, 900, 1000 consecutive nucleotides) of the RNA duplex are complementary or substantially complementary to a target mRNA that encodes a gene product involved in nicotine biosynthesis. Further aspects relate to a field or crop of tobacco plants comprising one or more of the constructs described herein. Still other aspects relate to a tobacco seed produced from one or more of the tobacco plants of the present invention.

Transgenic tobacco plants produced by the methods described herein can be cured by any of the tobacco curing techniques that are known in the art. As such, some embodiments relate to cured tobacco and cure tobacco products made from the transgenic plants described herein. In some embodiments, the cured tobacco product is a blended tobacco product. In some embodiments, the cured tobacco product is processed in a microbe-free environment. In other embodiments, the cured tobacco is contacted with sterilizing vapor, heat, or radiation so as to prevent the conversion of alkaloid to TSNAs.

Some aspects of the present invention relate to methods of preparing a tobacco cell having a reduced nicotine content, wherein the method comprises providing a tobacco cell with one or more interfering RNAs or one or more nucleic acid constructs encoding an interfering RNA comprising an RNA duplex, which comprises a first strand having a sequence substantially similar or identical to at least a portion of the coding sequence of a target gene and/or target gene product involved in nicotine and/or nornicotine biosynthesis, and a second strand that is complementary or substantially complementary to the first strand. In a preferred embodiment, the target gene product involved in nicotine biosynthesis is QPTase, PMTase, or A622.

Other aspects of the present invention relate to methods of preparing a tobacco plant having a reduced nicotine content comprising obtaining a tobacco cell in culture; providing to the tobacco cell one or more interfering RNAs or one or more nucleic acid constructs encoding an interfering RNA comprising an RNA duplex, which comprises a first strand having a sequence substantially similar or identical to at least a portion of the coding sequence of a target gene and/or target gene product involved in nicotine biosynthesis, and a second strand that is complementary or substantially complementary to the first strand; allowing expression of the interfering RNA, thereby reducing cellular nicotine content; and regenerating a tobacco plant from the tobacco cell. In some embodiments, the tobacco plants prepared by such method also have a reduced TSNA content, as compared to a conventional tobacco product of he same class, a reference tobacco product (e.g., IM16), or the same strain of tobacco prior to genetic modification.

Preparation of Preferred Transgenic Tobaccos

A first generation of transgenic Burley tobacco was created using a full-length antisense QPTase construct. Tobacco of the variety Burley 21 LA was transformed with the binary Agrobacterium vector pYTY32 to produce a low nicotine tobacco variety, Vector 21-41. The binary vector pYTY32 carried the 2.0 kb NtQPT1 root-cortex-specific promoter driving antisense expression of the NtQPT1 cDNA (SEQ. ID. NO. 1) and the nopaline synthase (nos) 3′ termination sequences from Agrobacterium tumefaciens T-DNA. The selectable marker for this construct was neomycin phosphotransferase (nptII) from E. coli Tn5 which confers resistance to kanamycin, and the expression nptII was directed by the nos promoter from Agrobacterium tumefaciens T-DNA. Transformed cells, tissues, and seedlings were selected by their ability to grow on Murashige-Skoog (MS) medium containing 300 μg/ml kanamycin. Burley 21 LA is a variety of Burley 21 with substantially reduced levels of nicotine as compared with Burley 21 (i.e., Burley 21 LA has 8% the nicotine levels of Burley 21, see Legg el al., Can J Genel Cylol, 13:287-91 (1971); Legg et al., J Hered, 60:213-17 (1969)).

One-hundred independent pYTY32 transformants of Burley 21 LA (T₀) were allowed to self. Progeny of the selfed plants (T₁) were germinated on medium containing kanamycin and the segregation of kanamycin resistance scored. T₁ progeny segregating 3:1 resulted from transformation at a single locus and were subjected to further analysis.

Nicotine levels of T₁ progeny segregating 3:1 were measured qualitatively using a micro-assay technique. Approximately ˜200 mg fresh tobacco leaves were collected and ground in 1 ml extraction solution (Extraction solution: 1 ml Acetic acid in 100 ml H₂O). Homogenate was centrifuged for 5 min at 14,000×g and supernatant removed to a clean tube, to which the following reagents were added: 100 μL NH₄OAC (5 g/100 ml H₂O+50 μL Brij 35); 500 μL Cyanogen Bromide (Sigma C-6388, 0.5 g/100 ml H₂O+50 μL Brij 35); 400 μL Aniline (0.3 ml buffered Aniline in 100 ml NH₄OAC+50 μL Brij 35). A nicotine standard stock solution of 10 mg/ml in extraction solution was prepared and diluted to create a standard series for calibration. Absorbance at 460 nm was read and nicotine content of test samples were determined using the standard calibration curve.

T₁ progeny that had less than 10% of the nicotine levels of the Burley 21 LA parent were allowed to self to produce T₂ progeny. Homozygous T₂ progeny were identified by germinating seeds on medium containing kanamycin and selecting clones in which 100% of the progeny were resistant to kanamycin (i.e., segregated 4:0; heterozygous progeny would segregate 3:1). Nicotine levels in homozygous and heterozygous T₂ progeny were qualitatively determined using the micro-assay and again showed levels less than 10% of the Burley 21 LA parent. Leaf samples of homozygous T₂ progeny were sent to the Southern Research and Testing Laboratory in Wilson, N.C. for quantitative analysis of nicotine levels using Gas Chromatography/Flame Ionization Detection (GC/FID). Homozygous T₂ progeny of transformant #41 gave the lowest nicotine levels (˜70 ppm), and this transformant was designated as “Vector 21-41.”

Vector 21-41 plants were allowed to self-cross, producing T₃ progeny. T₃ progeny were grown and nicotine levels assayed qualitatively and quantitatively. T₃ progeny were allowed to self-cross, producing T₄ progeny. Samples of the bulked seeds of the T₄ progeny were grown and nicotine levels tested.

In general, Vector 21-41 is similar to Burley 21 LA in all assessed characteristics, with the exception of alkaloid content and total reducing sugars (e.g., nicotine and nor-nicotine). Vector 21-41 may be distinguished from the parent Burley 21 LA by its substantially reduced content of nicotine, nor-nicotine and total alkaloids. As shown below, total alkaloid concentrations in Vector 21-41 are significantly reduced to approximately relative to the levels in the parent Burley 21 LA, and nicotine and nor-nicotine concentrations show dramatic reductions in Vector 21-41 as compared with Burley 21 LA. Vector 21-41 also has significantly higher levels of reducing sugars as compared with Burley 21 LA.

Field trials of Vector 21-41 T₄ progeny were performed at the Central Crops Research Station (Clayton, N.C.) and compared to the Burley 21 LA parent. The design was three treatments (Vector 21-41, a Burley 21 LA transformed line carrying only the NtQPT1 promoter [Promoter-Control], and untransformed Burley 21 LA [Wild-type]), 15 replicates, 10 plants per replicate. The following agronomic traits were measured and compared: days from transplant to flowering; height at flowering; leaf number at flowering; yield; percent nicotine; percent nor-nicotine; percent total nitrogen; and percent reducing sugars.

Vector 21-41 was also grown on approximately 5000 acres by greater than 600 farmers in five states (Pennsylvania, Mississippi, Louisiana, Iowa, and Illinois). The US Department of Agriculture, Agriculture Marketing Service (USDA-AMS) quantified nicotine levels (expressed as percent nicotine per dry weight) using the FTC method of 2,701 samples taken from these farms. Nicotine levels ranged from 0.01% to 0.57%. The average percent nicotine level for all these samples was 0.09%, with the median of 0.07%. Burley tobacco cultivars typically have nicotine levels between 2% and 4% dry weight (Tso, T. C., 1972, Physiology and Biochemistry of Tobacco Plants. Dowden, Hutchinson, and Ross, Inc. Stroudsbury).

A transgenic Flue-cured tobacco with a reduced amount of nicotine and TSNAs was created using an RNAi approach. FIG. 1 illustrates an RNAi construct that was used to create a reduced nicotine tobacco, wherein the root-specific promoter RD2 (Bp 1-2010) was used to drive expression of an RNAi cassette comprising an antisense full-length QPTase cDNA (Bp 2011-3409) linked to a 382 bp fragment of the cucumber aquaporin gene (Bp 3410-3792), which is linked to a sense full-length QPTase cDNA (Bp 3793-5191) and the GapC terminator (Bp5192-5688) (see SEQ. ID. No. 21). This first RNAi construct also comprises a GUS-selection cassette comprising the GapC promoter (Bp 1-1291), which drives expression of the GUS gene (Bp 1292-3103), linked to the GapC terminator (Bp 3104-3600) (see SEQ. ID. No. 24). This first RNAi construct was ligated into a binary vector, pBin19 which was then introduced into Agrobacterium tumefaciens. Leaf disks from flue-cured variety K326 were then transformed with Agrobacterium that contained the RNAi construct comprising the RNAi cassette and the GUS selection cassette. GUS-based selection was then employed to select positively transformed plantlets (buds), which were then regenerated to plants. Leaf samples were then harvested and the alkaloid content was then determined. The alkaloid content of samples obtained from some of the transgenic lines created with this first RNAi construct was 6000 ppm. Since the total alkaloid content in tobacco is about 90% nicotine, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 1 has reduced levels of nicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 2 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct has a QPTase inhibition cassette (SEQ. ID. No. 22) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the QPTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense fragment (360 bp) (Bp 2011-2370) of the QPTase gene, joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense fragment of the QPTase gene (360 bp) (Bp 3502-3861), which is joined to the GAD2 terminator (Bp 3862-4134). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSMI) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco was transformed with the construct shown in FIG. 2 using Agrobacterium-mediated transformation and 1,140 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 1,140 independent lines, 1,097 plants were harvested and tested for alkaloid content. A total of 608 lines were identified as having less than 1,000 ppm total alkaloid and 139 lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 2 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco was also transformed with the construct shown in FIG. 2 using Agrobacterium-mediated transformation and 385 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 385 independent lines, 350 lines of plants were harvested and tested for alkaloid content. A total of 142 lines were identified as having less than 1,000 ppm total alkaloid and 10 lines were identified as having less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Burley tobacco created using the construct shown in FIG. 2 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco was transformed with the construct shown in FIG. 2 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and 61 independent lines were selected, regenerated, and transplanted in the greenhouse. All 61 lines were tested for alkaloid content and a total of 10 lines were identified as having less than 1,500 ppm total alkaloids and a total of 3 lines were identified as having less than 1,000 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Oriental tobacco created using the construct shown in FIG. 2 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 3 illustrates another RNAi construct that can be used to create a reduced nicotine and TSNA transgenic tobacco. This RNAi construct has a PMTase inhibition cassette (SEQ. ID. No. 5) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the PMTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic acid (241 bp) (Bp 2011-2251) of a PMTase gene, joined to a FAD2 intron (Bp 2252-3382), which is joined to a sense nucleic acid of the PMTase gene (241 bp) (Bp 3383-3623), which is joined to the GAD2 terminator (Bp 3624-3896). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSMI) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco will be transformed with the construct shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Burley tobacco will be transformed with the construct shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated (see e.g., Broothaerts et al., Nature 433:629 (2005), herein expressly incorporated by reference in its entirety) or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 4 illustrates another RNAi construct that was used to create a reduced nicotine and TSNA transgenic tobacco. This RNAi construct has a A622 inhibition cassette (SEQ. ID. No. 3) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the A622 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic acid (628bp) (Bp 2011-2638) of the A622 gene, joined to a FAD2 intron (Bp 2639-3769), which is joined to a sense nucleic acid of the A622 gene (628bp) (Bp 3770-4397), which is joined to the GAD2 terminator (Bp 4398-4670). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco was transformed with the construct shown in FIG. 4 using Agrobacterium-mediated transformation and 270 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 270 independent lines, 259 plants were harvested and tested for alkaloid content. A total of 131 lines were identified as having less than 1,000 ppm total alkaloid and 45 lines were identified as having less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 4 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Several lines that were transformed with this construct were unexpectedly found to have conventional levels of nicotine but a significantly reduced amount of nornicotine. That is, 9 lines were found to have nicotine levels ranging from 2.17 mg/g to 3.99 mg/g and nornicotine levels less than or equal to 0.00 to 0.06 mg/g (see Table 2). TABLE 2 Transgenic tobacco having reduced nornicotine and conventional amounts of nicotine Alkaloid Nornicotine Nicotine new I.D (ppm) (mg/g) (mg/g) VDG 020 2486.53

2.30 VDG 032 4683.01

3.48 VDG 045 4490.79

3.94 VDG 052 2855.58

2.61 VDG 054 2291.89

2.17 VDG 077 4857.86

3.99 VDG 097 3072.40

2.58 VDG 107 4921.31

3.59 VDG 116 4960.64

3.56 Control-8 5005.22 0.28 4.02 Control-20 5711.97 0.34 5.35 Control-28 5196.25 0.24 4.52 *Highlighted entries show transgenic tobacco lines having a reduced amount of nornicotine and conventional amounts of nicotine. *Highlighted entries show transgenic tobacco lines having a reduced amount of nornicotine and conventional amounts of nicotine.

Tobacco products containing the selectively reduced nornicotine transgenic tobacco described above are also embodiments of the invention. That is, tobacco products comprising a transgenic tobacco that comprises (e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) a conventional amount of nicotine (e.g., at least, less than, greater than, or equal to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/g nicotine) and a reduced amount of nornicotine (e.g., 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification, are embodiments of the invention. Particularly preferred are transgenic tobacco and tobacco products made therefrom, which comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) a conventional amount of nicotine (e.g., at least, less than, greater than, or equal to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/g nicotine) and a reduced amount of nornicotine (e.g., 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification, and an isolated fragment of the A622 gene, in particular, comprising, consisting of, or consisting essentially of an isolated nucleic acid of SEQ. ID. No. 2, or the cassette of SEQ. ID. No. 3.

Burley tobacco will be transformed with the construct shown in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 4 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification. It is also expected that some lines of tobacco created with the afore-mentioned nucleic acid construct will retain conventional amounts of nicotine but will comprise a reduced amount of nornicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 4 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification. It is also expected that some lines of tobacco created with the afore-mentioned nucleic acid construct will retain conventional amounts of nicotine but will comprise a reduced amount of nornicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

FIG. 5 illustrates a double-knock-out RNAi construct, which has been created to develop a reduced nicotine and TSNA transgenic tobacco. This double-knock-out RNAi construct has a QPTase/A622 inhibition cassette (SEQ. ID. No.23) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the QPTase/A622 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to a QPTase antisense nucleic acid (360 bp) (Bp 2011-2370) of a QPTase gene, which is joined to a A622 antisense nucleic acid (628 bp) (Bp 2371-2998) of a A622 gene, which is joined to a FAD2 intron (Bp 2999-4129), which is joined to a sense nucleic acid of the A622 gene (628 bp) (Bp 4130-4757), which is joined to a sense nucleic acid of the QPTase gene (360 bp) (Bp 4758-5117), which is joined to the GAD2 terminator (Bp 5118-5390). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSMI) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco was transformed with the construct shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated, or biolistic transformation and 444 independent lines were selected, regenerated, and transplanted in the greenhouse. Each of these lines were analyzed for alkaloid content and 240 lines were found to have less than 1,000 ppm total alkaloid and 18 lines were found to have less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 5 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Significantly, for the first time, a plurality of different genes involved in nicotine biosynthesis have been inhibited using a single construct. This is particularly useful as it has been shown that genetic modification of genes in nicotine biosynthesis is leaky. That is, since there are several genes involved in nicotine biosynthesis, it is contemplated that the inhibition of one gene may be compensated for by other genes involved in the synthesis of nicotine. Accordingly, the inhibition of a plurality of genes in nicotine biosynthesis, as indicated by the present disclosure allows for a stronger control over the production of nicotine.

Burley tobacco will be transformed with the construct shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 5 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Oriental tobacco will also be transformed with the construct shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 5 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

It should be emphasized that other promoters and terminators can be used with the nucleic acids of the invention interchangeably. Although RD2 (SEQ. ID. No. 10) is a preferred root-specific promoter, there are other root-specific promoters that can be used, as well. For example, the putrescene methyl transferase 1 promoter (PMT-1) (SEQ. ID. No. 11) is a root-specific promoter that can be used in place of the RD2 promoter in any of the constructs described above. Similarly, although the actin2 promoter (SEQ. ID. No. 13) is preferred for driving expression of a norflurazone resistance gene, other constitutive promoters such as the GapC promoter (SEQ. ID. No. 12), the tobacco alcohol dehydrogenase (ADP) (SEQ. ID. No. 14) and the Arabidopsis ribosomal protein L2 (RPL2P) (SEQ. ID. No. 15) can be used to drive expression of the norflurazone resistance gene. Additionally, developmentally regulated promoters such as, cinnamyl alcohol dehydrogenase (SEQ. ID. No. 16) and metallothionein I promoter (SEQ. ID. No. 17) can be used interchangeable with the cassettes described herein.

Further, in some embodiments, a plurality of constitutive promoters, in tandem, can be used to drive expression of the norflurazone resistance gene. Additionally, a plurality of root-specific promoters can be used to drive expression one or more of the inhibition cassettes described above (e.g., the QPTase inhibition cassette, the PMTase inhibition cassette, the A622 inhibition cassette, or a double-knockout inhibition cassette). Developmentally regulated promoters, a plurality of developmentally regulated promoters, constitutive promoters, or a plurality of constitutive promoters can also be used to drive expression of one or more of the inhibition or selection cassettes described above. Accordingly, any promoter operable in tobacco can be used to drive expression of any of the inhibition cassettes or the selection cassette described herein (e.g., nos, 35S, or CAMV). Terminators, such as GAD2 terminator (SEQ. ID. No. 18) and the FAD 2 (SEQ. ID. No. 19) or PAP1 introns can be used interchangeably, as well.

Other aspects of the invention concern the discovery of several mutants of the phytoene desaturase gene that confer resistance to the herbicide norflurazone (e.g., SEQ. ID. Nos.7, 8, and 9). These herbicide resistance genes were used as selectable markers in the transformations above. Typically, the selection was accomplished by introducing the transformed plant tissue to the norflurazone (e.g., 0.005 μM-0.1 μMconc.). That is, the concentration of norflurazone that can be used to select positive transformants containing a norflurazone resistance gene, as described herein can be at least, less than, greater than, or equal to 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μM. Preferably, less than or equal to 0.05 μM concentration of norflurazone is used when selecting transformants with Flue-cured tobacco and less than or equal to 0.0125 μM concentration norflurazone is used when selecting transformants with Burley tobacco. As the plantlet develops, selection was accomplished by differentiating the green shoots (positive transformants) from the yellow or white shoots (negative transformants). Once selection was made, the herbicide was removed and the plantlet was allowed to develop in the greenhouse.

The norflurazone resistant phytoene desaturase mutants (PDSM-1, PDSM-2, and PDSM-3) were generated by site-directed mutagenesis of particular regions of the gene believed to be involved in binding of the herbicide. Constructs carrying the various PDSM genes were then transferred to tobacco leaf disks by conventional Agrobacterium transformation and the resistance to norflurazone was analyzed at various concentrations. After several iterations, the mutants described as SEQ. ID. Nos. 7, 8, and 9, were identified as sequences that confer resistance to norflurazone. Accordingly, aspects of the invention concern the PDSM genes described herein, their use in plants as selectable markers to identify plant cells that contain a transformed gene, whether in tissue culture or in the field, and methods of identifying new PDSM genes that confer norflurazone resistance.

In a first selection construct, the Arabidopsis phytoene desaturase gene (PDS) (SEQ. ID. No. 26) was mutated using site-directed mutagenesis, such that a T to G mutation at position 1478, resulting in a Valine to Glycine change at amino acid residue 493 was created. To generate the norflurazone resistance gene, the open reading frame of the Arabidopsis phytoene desaturase gene was amplified and cloned into the TOPO vector (Invitrogen). A single base pair change from T-G at nucleotide position 1478, leading to a Valine to Glycine change at amino acid residue 493, was introduced using QuickChange Site-directed Mutagenisis Kit (Stratgene). The point mutation was verified by sequencing and the resultant mutant was named PDSM-1 (SEQ. ID. No. 7). The 1.729 Kb PDSM1 sequence was then amplified and ligated into the binary vector pWJ001, a pCambia derivative that contained the RNAi cassettes above, which was then introduced into Agrobacterium tumefaciens. A similar approach was used to generate the PDSM-2 and PDSM-3 mutants described in the sequence listing as SEQ. ID. NOs.8 and 9.

That is, in a second selection construct, the Arabidopsis phytoene desaturase gene (PDS) (SEQ. ID. No. 26) was mutated using site-directed mutagenesis, such that a G to C mutation at position 863, resulting in a Arginine to Proline change at amino acid residue 288 was created. To generate the norflurazone resistance gene, the open reading frame of the Arabidopsis phytoene desaturase gene was amplified and cloned into the TOPO vector (Invitrogen). A single base pair change was introduced using QuickChange Site-directed Mutagenisis Kit (Stratgene). The point mutation was verified by sequencing and the resultant mutant was named PDSM-2. The 1.729 Kb PDSM-2 sequence was then amplified and ligated into the binary vector pWJ001, a pCambia derivative that contained the RNAi cassettes above, which was then introduced into Agrobacterium tumefaciens

Further, in a third selection construct, the Arabidopsis phytoene desaturase gene (PDS) (SEQ. ID. No. 26) was mutated using site-directed mutagenesis, such that a T to C mutation at position 1226, resulting in a Leucine to Proline change at amino acid residue 409 was created. To generate the norflurazone resistance gene, the open reading frame of the Arabidopsis phytoene desaturase gene was amplified and cloned into the TOPO vector (Invitrogen). A single base pair change was introduced using QuickChange Site-directed Mutagenisis Kit (Stratgene). The point mutation was verified by sequencing and the resultant mutant was named PDSM-3. The 1.729 Kb PDSM-2 sequence was then amplified and ligated into the binary vector pWJ001, a pCambia derivative that contained the RNAi cassettes above, which was then introduced into Agrobacterium tumefaciens

Accordingly, aspects of the invention concern methods of identifying a mutation on a phytoene desaturase gene that confers resistance to an herbicide, preferably norflurazone. By one approach, a phytoene desaturase gene is provided, preferably SEQ. ID. No. 26, a nucleotide in said gene is mutated so as to generate a mutant phytoene desaturase gene, said mutant phytoene desaturase gene is transformed to a plant cell so as to generate a plant cell comprising said mutant phytoene desaturase gene, said plant cell comprising said mutant phytoene desaturase gene is then contacted with an herbicide, preferably norflurazone, and the presence or absence of a resistance to said herbicide is identified, whereby the presence of a resistance to said herbicide identifies said mutation as one that confers resistance to said herbicide. By one approach, the entire sequence of a phytoene desaturase gene (e.g., SEQ. ID. NO. 26) is mutated one residue at a time and each mutant is screened for resistance to the herbicide. Accordingly, aspects of the invention include compositions (e.g., nucleic acid constructs or cassettes, plant cells, plants, tobacco, or tobacco products) that comprise, consist, consist essentially of a mutant phytoene desaturase nucleic acid of SEQ. ID. NO. 7, 8, or 9 or fragment thereof at least or equal to 30, 50, 100, 200, 400, 500, 700, 900, 1000, 1200, 1400, 1600, or 1700 consecutive nucleotides of in length that confers resistance to an herbicide, in particular norflurazone. Aspects of the invention also include compositions (e.g., nucleic acid constructs or cassettes, plant cells, plants, tobacco, or tobacco products) comprising the mutant phytoene desaturase protein or fragments thereof (e.g., at least 15, 25, 50, 100, 200, 300, 400, 500 consecutive amino acids of a protein encoded by SEQ. ID. Nos. 7, 8, or 9) that confer resistance to an herbicide, in particular norflurazone.

The nucleic acid sequences, cassettes, and constructs described herein can also be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence can be used in some embodiments of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of the nucleic acid embodiments described herein that complement said sequences and have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. In some contexts, the phrase “substantial sequence similarity” in the present specification and claims means that DNA, RNA or amino acid sequences which have slight and non-consequential sequence variations from the actual sequences disclosed and claimed herein are considered to be equivalent to the sequences of the present invention. In this regard, “slight and non-consequential sequence variations” mean that “similar” sequences (i.e., the sequences that have substantial sequence similarity with the DNA, RNA, or proteins disclosed and claimed herein) will be functionally equivalent to the sequences disclosed and claimed in the present invention. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein.

Additional nucleic acid embodiments include sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to the nucleic acids, nucleic acid constructs, and nucleic acid cassettes provided herein. Preferably these sequences also perform the functions of the particular nucleic acid embodiment (e.g., inhibition of nicotine or nornicotine production or confer resistance to norflurazone). Determinations of sequence similarity are made with the two sequences aligned for maximum matching; gaps in either of the two sequences being matched are allowed in maximizing matching. Gap lengths of 10 or less are preferred, gap lengths of 5 or less are more preferred, and gap lengths of 2 or less still more preferred.

Additional nucleic acid embodiments also include nucleic acids that hybridize to the nucleic acid sequences disclosed herein under low, medium, and high stringency, wherein said additional nucleic acid embodiments also perform the function of the particular embodiment (e.g., inhibit nicotine or nornicotine production or confer resistance to norflurazone). Identification of nucleic acids that hybridize to the embodiments described herein can be determined in a routine manner. (See J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory)). For example, hybridization of such sequences may be carried out under conditions of reduced stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 0.3 M NaCl, 0.03 M sodium citrate. 0.1% SDS at 60 degrees C., or even 70 degrees C.). Preferably these sequences also perform the functions of the particular nucleic acid embodiment (e.g., inhibition of nicotine or nornicotine production or confer resistance to norflurazone).

The examples described herein demonstrate that several different RNAi constructs can be used to effectively reduce the levels of nicotine, and/or nornicotine in tobacco. Additionally, these examples demonstrate that several mutant phytoene desaturase genes, which confer resistance to the herbicide norflurazone, have been created and that selection cassettes comprising these herbicide resistant nucleic acids can be used to determine the presence of a linked gene in transformed tobacco cells. The section below describes typical curing methods which may be used to prepare the tobacco once it is harvested.

Curing

The curing process, which typically lasts about 1 week, brings out the flavor and aroma of tobacco. Several methods for curing tobacco may be used, and indeed many methods have been previously disclosed. For example, U.S. Pat. No. 4,499,911 to Johnson; U.S. Pat. No. 5,685,710 to Martinez Sagrera; U.S. Pat. No. 3,905,123 to Fowler; U.S. Pat. No. 3,840,025 to Fowler; and U.S. Pat. No. 4,192,323 to Home, (each hereby expressly incorporated by reference in its entirety) describe aspects of the tobacco curing process which may be used for some embodiments of the present invention. Conventionally, “sticks” that are loaded with tobacco are placed into bulk containers and placed into closed buildings having a heat source known as a curing barn. A flue is often used to control the smoke (thus earning the term “flue-cured”). The method of curing will depend, in some cases, on the type of tobacco-use cessation product desired, (i.e., snuff, cigarettes, or pipe tobacco may preferably utilize different curing methods) and preferred methods may vary from region to region and in different countries. In some approaches, the stems and midveins of the leaf are removed from the leaves prior to curing to yield a high quality, low nitrosamine tobacco product.

“Flue curing” is a popular method for curing tobacco in Virginia, North Carolina, and the Coastal Plains regions of the United States. This method is used mainly in the manufacture of cigarettes. Flue curing requires a closed building equipped with a system of ventilation and a source of heat. The heating can be direct or indirect (e.g., radiant heat). When heat and humidity are controlled, leaf color changes, moisture is quickly removed, and the leaf and stems dry. Careful monitoring of the heating and humidity can reduce the accumulation of nitrosamines.

Another curing method is termed “air curing”. In this method, an open framework is prepared in which sticks of leaves (or whole plants) are hung so as to be protected from both wind and sun. Leaf color changes from green to yellow, as leaves and stems dry slowly.

“Fire curing” employs an enclosed barn similar to that used for flue curing. The tobacco is hung over low temperature fire so that the leaves cure in a smoke-laden atmosphere. This process uses lower temperatures, so the process may take up to a month, in contrast to flue curing, which takes about 6 to 8 days.

A further curing method, termed “sun curing” is the drying of uncovered sticks or strings of tobacco leaves in the sun. The best known sun-cured tobaccos are the so-called oriental tobaccos of Turkey, Greece, Yugoslavia, and nearby countries.

The curing process, and most particularly the flue-curing process, is generally divided into the following four stages:

A) Firing Up: During this step, the tobacco leaves turn bright lemon-orange in color. This is achieved by a gradual increase in temperature.

B) Leaf Yellowing: In this step any moisture is removed. This creates the “yellowing” of the tobacco. It also prepares the tobacco for drying in the next step.

C) Leaf Drying: Leaf drying, an important step in the curing process, requires much time for the tobacco to dry properly. Additionally, air flow is increased in this step to facilitate the drying process.

D) Stem Drying: The drying process continues, as the stem of the tobacco leaf becomes dried.

The cured tobacco may then be blended with other tobaccos or other materials to create the product to be used for the tobacco-use cessation method. The section below describes typical methods of blending and preparing the tobacco product.

Making Tobacco Cessation Products by Tobacco Blending and/or Addition of Exogenous Nicotine

In some embodiments, a tobacco comprising a reduced amount of alkaloid (e.g., a reduced amount of nicotine, nornicotine, and/or TSNAs) is contacted with an exogenous nicotine so as to raise the level of nicotine in the contacted tobacco in a controlled fashion. By this approach, nicotine levels in tobacco that comprises a reduced amount of endogenous nicotine (i.e., nicotine that is produced by the plant from which the tobacco is obtained) can be selectively raised to levels that are commensurate with conventional full-flavor cigarettes, light cigarettes, or ultra-light cigarettes. (See e.g., WO 2005/018307, which designates the United States and was published in English, herein expressly incorporated by reference in its entirety). For example, tobacco comprising a reduced amount of endogenous nicotine and/or TSNAs can be contacted with an amount of exogenous nicotine that is at least, equal to, or more than 0.3 mg/g-20.0 mg/g (nicotine/gram of tobacco). That is, tobacco comprising a reduced amount of endogenous nicotine and/or TSNAs can be contacted with an amount of exogenous nicotine that is at least, equal to, or more than 0.3 mg/g, 0.4 mg/g, 0.5 mg/g, 0.6 mg/g, 0.7 mg/g, 0.8 mg/g, 0.9 mg/g, 1.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g, 1.6 mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, 2.0 mg/g, 2.1 mg/g, 2.2 mg/g, 2.3 mg/g, 2.4 mg/g, 2.5 mg/g, 2.6 mg/g, 2.7 mg/g, 2.8 mg/g, 2.9 mg/g, 3.0 mg/g, 3.1 mg/g, 3.2 mg/g, 3.3 mg/g, 3.4 mg/g, 3.5 mg/g, 3.6 mg/g, 3.7 mg/g, 3.8 mg/g, 3.9 mg/g, 4.0 mg/g, 4.1 mg/g, 4.2 mg/g, 4.3 mg/g, 4.4 mg/g, 4.5 mg/g, 4.6 mg/g, 4.7 mg/g, 4.8 mg/g, 4.9 mg/g, 5.0 mg/g, 5.1 mg/g, 5.2 mg/g, 5.3 mg/g, 5.4 mg/g, 5.5 mg/g, 5.6 mg/g, 5.7 mg/g, 5.8 mg/g, 5.9 mg/g, 6.0 mg/g, 6.1 mg/g, 6.2 mg/g, 6.3 mg/g, 6.4 mg/g, 6.5 mg/g, 6.6 mg/g, 6.7 mg/g, 6.8 mg/g, 6.9 mg/g, 7.0 mg/g, 7.1 mg/g, 7.2 mg/g, 7.3 mg/g, 7.4 mg/g, 7.5 mg/g, 7.6 mg/g, 7.7 mg/g, 7.8 mg/g, 7.9 mg/g, 8.0 mg/g, 8.1 mg/g, 8.2 mg/g, 8.3 mg/g, 8.4 mg/g, 8.5 mg/g, 8.6 mg/g, 8.7 mg/g, 8.8 mg/g, 8.9 mg/g, 9.0 mg/g, 9.1 mg/g, 9.2 mg/g, 9.3 mg/g, 9.4 mg/g, 9.5 mg/g, 9.6 mg/g, 9.7 mg/g, 9.8 mg/g, 9.9 mg/g, 10.0 mg/g, 10.1 mg/g, 10.2 mg/g, 10.3 mg/g, 10.4 mg/g, 10.5 mg/g, 10.6 mg/g, 10.7 mg/g, 10.8 mg/g, 10.9 mg/g, 11.0 mg/g, 11.1 mg/g, 11.2 mg/g, 11.3 mg/g, 11.4 mg/g, 11.5 mg/g, 11.6 mg/g, 11.7 mg/g, 11.8 mg/g, 11.9 mg/g, 12.0 mg/g, 12.1 mg/g, 12.2 mg/g, 12.3 mg/g, 12.4 mg/g, 12.5 mg/g, 12.6 mg/g, 12.7 mg/g, 12.8 mg/g, 12.9 mg/g, 13.0 mg/g, 13.1 mg/g, 13.2 mg/g, 13.3 mg/g, 13.4 mg/g, 13.5 mg/g. 13.6 mg/g, 13.7 mg/g, 13.8 mg/g, 13.9 mg/g, 14.0 mg/g, 14.1 mg/g, 14.2 mg/g, 14.3 mg/g, 14.4 mg/g, 14.5 mg/g, 14.6 mg/g, 14.7 mg/g, 14.8 mg/g, 14.9 mg/g, 15.0 mg/g, 15.1 mg/g, 15.2 mg/g, 15.3 mg/g, 15.4 mg/g, 15.5 mg/g, 15.6 mg/g, 15.7 mg/g, 15.8 mg/g, 15.9 mg/g, 16.0 mg/g, 16.1 mg/g, 16.2 mg/g, 16.3 mg/g, 16.4 mg/g, 16.5 mg/g, 16.6 mg/g, 16.7 mg/g, 16.8 mg/g, 16.9 mg/g, 17.0 mg/g, 17.1 mg/g, 17.2 mg/g, 17.3 mg/g, 17.4 mg/g, 17.5 mg/g, 17.6 mg/g, 17.7 mg/g, 17.8 mg/g, 17.9 mg/g, 18.0 mg/g, 18.1 mg/g, 18.2 mg/g, 18.3 mg/g, 18.4 mg/g, 18.5 mg/g, 18.6 mg/g, 18.7 mg/g, 18.8 mg/g, 18.9 mg/g, 19.0 mg/g, 19.1 mg/g, 19.2 mg/g, 19.3 mg/g, 19.4 mg/g, 19.5 mg/g, 19.6 mg/g, 19.7 mg/g, 19.8 mg/g, 19.9 mg/g, and 20.0 mg/g (nicotine/gram tobacco)). In some of the aforementioned embodiments, said tobacco is genetically modified and comprises one or more of the isolated nucleic acids, isolated nucleic acid cassettes, or isolated nucleic acid constructs, described herein.

Nicotine-containing fractions, nicotine, or nicotine salts of organic acids are added to the reduced-nicotine transgenic tobacco by contacting said tobacco (e.g., spraying or additive application), with or without propylene glycol, solvent, flavoring, or water at any stage of the harvesting, curing, fermenting, aging, reconstituting, expanding, or otherwise processing of the tobacco, preferably at a stage that is post-cure, when flavorings and additives are provided. By “exogenous nicotine” is meant nicotine, nicotine derivatives, nicotine analogs, nicotine-containing fractions (e.g., extracts of Nicotiana), and nicotine salts of organic acids obtained from a source outside of the transgenic tobacco to which the exogenous nicotine is applied. In this manner, a transgenic tobacco with virtually any amount of nicotine can be obtained.

In some embodiments, the exogenous nicotine (e.g., commercially available nicotine salts, liquid, or a nicotine-containing extract prepared from a Nicotiana plant or portion thereof) is contacted with a reduced-alkaloid transgenic tobacco (e.g., a transgenic tobacco comprising a reduced amount of nicotine and/or TSNA as prepared as described herein) after the transgenic tobacco has been made substantially free of microbes (e.g., bacteria, yeast, mold, or fungi). The reduced alkaloid transgenic tobacco can be made substantially-free of microbes (e.g., an aseptic preparation) by employing sterilization, heat treatment, pasteurization, steam treatment, gas treatment, and radiation (e.g., gamma, microwave, and ultraviolet). The term “substantially-free of microbes” in some contexts can mean an amount of bacteria, mold, fungi, or yeast that is reduced to the point that the conversion of nicotine or total alkaloid to TSNA is negligible, for example, a tobacco product comprises (e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods)a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 μg/g, 4.0 μg/g, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, or 0.5 μg/g) after prolonged storage (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years)). The term “substantially-free of microbes” also includes the term “substantially-free of bacteria,” which means in some contexts that the tobacco or tobacco product is substantially-free of Arthrobacter, Proteus, nicotine oxidizing bacteria, such as P-34, Psuedomonas, Xantomonas, or Zoogloea strains of bacteria For example, a tobacco or tobacco product is substantially-free of bacteria or a particular strain of bacteria when said tobacco or tobacco product has less than or equal to 20% of the bacteria or a specific strain of bacteria normally present on the tobacco or tobacco product in the absence of application of a technique to rid the tobacco or tobacco product of bacteria (e.g. less than or equal to 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). With respect to transgenic tobacco described herein, the term “substantially-free of bacteria” can refer to tobacco or a tobacco product containing said transgenic tobacco that has less than or equal to 20% of the bacteria normally present on the strain of tobacco prior to genetic modification and/or application of a technique to rid the tobacco or tobacco product of bacteria (e.g., less than or equal to 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%).

Once the exogenous nicotine has been contacted with the microbe-free tobacco, it is preferably processed and packaged aseptically and the tobacco product is maintained in an airtight container so as to not re-introduce microbes that convert the exogenous nicotine to TSNAs. By using the aseptic processing, manufacturing, and packaging procedures, described herein, one can maintain an amount of total TSNA (e.g., the collective content of NNN, NAT, NAB, and NNK) in a commercially available tobacco product, which comprises exogenous nicotine, such that said product comprises (e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) an mount that is less than or equal to 5.0 μg/g (e.g., less than or equal to 5.0 μg/g, 4.0 μg, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, or 0.05 μg/g) for at least 1 week, 1 month, or 1-5 years after packaging (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years). In some embodiments, the exogenous nicotine is contacted with a tobacco comprising one of the nucleic acid constructs described herein and comprising (e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) a collective content of NNN, NAT, NAB, and NNN that is less than or equal to 2.0 μg/g (e.g., less than or equal to 2.0 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, 0.3 μg/g, or 0.1 μg/g). In some embodiments, an amount on the leaf or tobacco rod or the delivery (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) is maintained to a level such that a collective content of NNN, NAT, NAB, and NNN of less than or equal to 5.0 μg/g (e.g., less than or equal to 5.0 μg/g, 4.0 μg, 3.0 μg/g, 2.0 μg/g, 1.0 μg/g, or 0.05 μg/g) in a tobacco product containing said tobacco can be maintained for at least at least 1 week, 1 month, or 1-5 years after packaging (e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0 years). Accordingly, several embodiments address the problem of gradually increasing TSNA levels in alkaloid-containing tobacco products by employing processing, storage, and packaging methods that reduce the amount of microbial flora on the tobacco, limit the re-introduction of microbes during processing and maintain a reduced amount of microbes (e.g., bacteria) once the product is packaged, stored, and sold. Tobacco and tobacco products comprising tobacco having a reduced amount of endogenous nicotine and an amount of exogenous nicotine can be analyzed by various methods to confirm that said tobacco and said tobacco products are “reduced risk” or have less of a potential to contribute to a tobacco-related disease, as compared to the parent strain of tobacco having conventional amounts of endogenous nicotine or a reference tobacco.

It addition, it may be desirable to blend tobacco of varying nicotine levels to create the cessation product having the desired level of nicotine. This blending process is typically performed after the curing process, and may be performed by conventional methods. Preferred tobacco blending approaches are provided in Examples 19 and 20. In some embodiments, blending of the transgenic tobacco is conducted to prepare the tobacco so that it will contain specific amounts of nicotine and/or TSNA in specific products. Preferably, the blending is conducted so that tobacco products of varying amounts of nicotine and/or TSNAs are made in specific products.

A mixture that contains different types of tobacco is desirably substantially homogeneous throughout in order to avoid undesirable fluctuations in taste or nicotine levels. Typically, tobacco to be blended may have a moisture content between 30 and 75%. As an example, the tobacco is first cut or shredded to a suitable size, then mixed in a mixing device, such as a rotating drum or a blending box. One such known mixing device is a tumbling apparatus that typically comprises a rotating housing enclosing mixing paddles which are attached to and, therefore, rotate with the housing to stir the tobacco components together in a tumbling action as the drum turns.

After the desired tobaccos are thoroughly mixed, the resulting tobacco blend is removed from the mixing apparatus and bulked to provide a continuous, generally uniform quantity of the tobacco blend. The tobacco is then allowed to remain relatively undisturbed (termed the “bulking step”) for the required period of time before subsequent operations are performed. The bulking step typically takes 30 minutes or less, and may be carried out on a conveyor belt. The conveyor belt allows the blended tobacco to remain in bulk form in an undisturbed condition while it is continuously moving the tobacco blend through the process from the mixing stage to the expansion stage.

The tobacco blend is typically expanded by the application of steam. The tobacco mixture is typically subjected to at least 0.25 pounds of saturated steam at atmospheric conditions per pound of blended tobacco for at least 10 seconds to provide an increase in moisture of at least 2 weight percent to the tobacco blend. After the tobacco blend has been expanded, it is dried. A typical drying apparatus uses heated air or superheated steam to dry the tobacco as the tobacco is conveyed by the heated air or steam stream through a drying chamber or series of drying chambers. Generally, the wet bulb temperature of the drying air may be from about 150 degrees F. to about 211 degrees F. The tobacco blend is typically dried to a moisture content of from about 60 percent to about 5 percent. The dried, expanded tobacco blend is then in a suitable mode to be processed into the tobacco-use cessation product as described below.

Some blending approaches begin with tobacco prepared from varieties that have extremely low amounts of nicotine, nornicotine, and/or TSNAs. By blending prepared tobacco from a low nicotine/TSNA variety (e.g., undetectable levels of nicotine and/or TSNAs ) with a conventional tobacco (e.g., Burley, which has 30,000 parts per million (ppm) nicotine and 8,000 parts per billion (ppb) TSNA; Flue-Cured, which has 20,000 ppm nicotine and 300 ppb TSNA; and Oriental, which has 10,000 ppm nicotine and 100 ppb TSNA), tobacco products having virtually any desired amount of nicotine and/or TSNAs can be manufactured. Other approaches blend only low nicotine/TSNA tobaccos (e.g., genetically modified Burley, genetically modified Virginia flue, and genetically modified Oriental tobaccos that contain reduced amounts of nicotine and/or TSNAs). Tobacco products having various amounts of nicotine and/or TSNAs can be incorporated into tobacco-use cessation kits and programs to help tobacco users reduce or eliminate their dependence on nicotine and reduce the carcinogenic potential.

By one approach, a step 1 tobacco product is comprised of approximately 25% low nicotine/TSNA tobacco and 75% conventional tobacco; a step 2 tobacco product can be comprised of approximately 50% low nicotine/TSNA tobacco and 50% conventional tobacco; a step 3 tobacco product can be comprised of approximately 75% low nicotine/TSNA tobacco and 25% conventional tobacco; and a step 4 tobacco product can be comprised of approximately 100% low nicotine/TSNA tobacco and 0% conventional tobacco. A tobacco-use cessation or nicotine and/or TSNA reduction kit can comprise an amount of tobacco product from each of the aforementioned blends to satisfy a consumer for a single month program. That is, if the consumer is a one pack per day smoker, for example, a single month kit would provide 7 packs from each step, a total of 28 packs of cigarettes. Each tobacco-use cessation kit would include a set of instructions that specifically guide the consumer through the step-by-step process. Of course, tobacco products having specific amounts of nicotine and/or TSNAs would be made available in conveniently sized amounts (e.g., boxes of cigars, packs of cigarettes, tins of snuff and pouches or twists of chew) so that consumers could select the amount of nicotine and/or TSNA they individually desire. There are many ways to obtain various low nicotine/low TSNA tobacco blends using the teachings described herein and the following is intended merely to guide one of skill in the art to one possible approach.

To obtain a step 1 tobacco product, which is a 25% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 25%/75% ratio respectively to obtain a Burley tobacco product having 22,500 ppm nicotine and 6,000 ppb TSNA, a Flue-cured product having 15,000 ppm nicotine and 225 ppb TSNA, and an Oriental product having 7,500 ppm nicotine and 75 ppb TSNA. Similarly, to obtain a step 2 product, which is 50% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 50%/50% ratio respectively to obtain a Burley tobacco product having 15,000 ppm nicotine and 4,000 ppb TSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb TSNA, and an Oriental product having 5000 ppm nicotine and 50 ppb TSNA. Further, a step 3 product, which is a 75%/25% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 75%/25% ratio respectively to obtain a Burley tobacco product having 7,500 ppm nicotine and 2,000 ppb TSNA, a Flue-cured product having 5,000 ppm nicotine and 75 ppb TSNA, and an Oriental product having 2,500 ppm nicotine and 25 ppb TSNA.

By a preferred method, conventional Virginia flue tobacco was blended with genetically modified Burley (i.e., Burley containing a significantly reduced amount of nicotine and nitrosamine) to yield a blended tobacco that was incorporated into three levels of reduced nicotine cigarettes (delivery in main-stream smoke by the FTC method): a step 1 cigarette containing 0.6 mg nicotine, a step 2 cigarette containing 0.3 mg nicotine, and a step 3 cigarette containing less than 0.05 mg nicotine. The amount of total TSNA was found to range between approximately 0.17 μg/g-0.6 μg/g (delivery in main-stream smoke by the FTC method).

In some cigarettes, approximately, 28% of the blend was Virginia flue tobacco, approximately 29% of the blend was genetically modified (i.e., reduced nicotine Burley), approximately 14% of the blend was Oriental, approximately 17% of the blend was expanded flue-cured stem, and approximately 12% was standard commercial reconstituted tobacco. The amount of total TSNAs in cigarettes containing this blend was approximately 1.5 μg/g (delivery in main-stream smoke by the FTC method).

It should be appreciated that tobacco products are often a blend of many different types of tobaccos, which were grown in many different parts of the world under various growing conditions. As a result, the amount of nicotine and TSNAs will differ from crop to crop. Nevertheless, by using conventional techniques one can easily determine an average amount of nicotine and TSNA per crop used to create a desired blend. It should also be appreciated that reconstituted, expanded, chemically treated, or microbial treated tobacco can be blended with the transgenic tobacco described herein. By adjusting the amount of each type of tobacco that makes up the blend one of skill can balance the amount of nicotine and/or TSNA with other considerations such as appearance, flavor, and smokability. In this manner, a variety of types of tobacco products having varying level of nicotine and/or nitrosamine, as well as, appearance, flavor and smokability can be created.

Furthermore, it should also be appreciated that the blending steps described above are not necessary for all embodiments. That is, a tobacco product within the scope of the present invention can be made by using the reduced nicotine tobacco described herein without blending with another type of tobacco.

Nicotine Reduction and/or Tobacco-Use Cessation Programs Methods

The methods described herein facilitate nicotine reduction, TSNA reduction, and/or tobacco-use cessation by allowing the individual to retain the secondary factors of addiction such as smoke intake, oral fixation, and taste, while reducing the addictive nicotine levels consumed. Eventually, complete cessation is made possible because the presence of addiction for nicotine is decreased while the individual is allowed to maintain dependence on the secondary factors addiction as described above.

As mentioned above, embodiments include tobacco products that have been carefully blended so that desired levels of nicotine and/or TSNAs are obtained. For example, tobacco having a reduced level of nicotine and/or TSNAs, prepared as described above, can be blended with conventional tobacco so as to obtain virtually any amount of nicotine. Additionally, as mentioned above, exogenous nicotine can be added to the tobacco or tobacco product. Further, two or more varieties of tobacco (e.g., transgenic reduced alkaloid Burley, transgenic reduced alkaloid Flue-cured, and/or transgenic reduced alkaloid Oriental) can be blended so as to achieve a desired taste while maintaining nicotine levels at less than 7,000 ppm, 5,000 ppm, 3000 ppm, 2000 ppm, 1000 ppm, or 500 ppm and TSNA levels at 0.5 μg/g or less (delivery in main-stream smoke by the FTC method). In this manner, differences in amounts of nicotine and/or TSNAs can be incrementally adjusted.

In some embodiments, a stepwise nicotine and/or TSNA reduction and/or tobacco-use cessation program can be established using the reduced nicotine and/or TSNA products described above. As an example, the program participant can be identified as an individual that desires a reduction in the consumption of nicotine and/or TSNAs or as an individual that desires cessation of tobacco use. The program participant may, optionally, determine his or her current level of nicotine intake. The program participant then begins the program at step 1, with a tobacco product having a reduced amount of nicotine and/or TSNAs, as compared to the tobacco product that was used prior to beginning the program. After a period of time, the program participant proceeds to step 2, using a tobacco product with less nicotine and/or TSNAs than the products used in step 1. The program participant, after another period of time, reaches step 3, wherein the program participant begins using a tobacco product with less nicotine than the products in step 2, and so on. Ultimately, the program participant uses a tobacco product having an amount of nicotine and/or TSNAs that is less than that which is sufficient to become addictive or to maintain an addiction. Thus, the nicotine reduction and/or tobacco-use cessation program limits the exposure of a program participant to nicotine and/or TSNAs and, concomitantly, the harmful effect of nicotine, yet retains the secondary factors of addiction, including but not limited to, smoke intake, oral fixation, and taste.

For example, (with reference to the delivery of nicotine in main-stream smoke by the FTC method) a smoker can begin the program smoking cigarettes having 5 mg of nicotine, move to smoking cigarettes with 3 mg of nicotine, followed by cigarettes having 1 mg nicotine, followed by cigarettes having 0.5 mg nicotine, followed by cigarettes having less than 0.1 mg nicotine until the consumer decides to smoke only the cigarettes having virtually no nicotine and nitrosamines or quitting smoking altogether. Preferably, a three-step program is followed whereby at step 1, cigarettes containing 0.6 mg nicotine are used; at step 2, cigarettes containing 0.3 mg nicotine are used; and at step 3, cigarettes containing less than 0.1 mg nicotine are used. More preferably, a three-step program is followed whereby at step 1, cigarettes containing 0.6 mg nicotine are used; at step 2, cigarettes containing 0.3 mg nicotine are used; and at step 3, cigarettes containing less than 0.05 mg nicotine used. Accordingly, the reduced nicotine and/or TSNA products described herein provide the basis for an approach to reduce the carcinogenic potential in a human in a step-wise fashion.

In another example, (with reference to the delivery of nicotine in main-stream smoke by the FTC method) a smoker can begin the program smoking cigarettes having 5 mg of nicotine and 1.5 μg of nitrosamine, move to smoking cigarettes with 3 mg of nicotine and 1 μg of nitrosamine, followed by cigarettes having 1 mg nicotine and 0.5 μg nitrosamine, followed by cigarettes having 0.5 mg nicotine and 0.25 μg nitrosamine, followed by cigarettes having less than 0.1 mg nicotine and less than 0.1 μg TSNA until the consumer decides to smoke only the cigarettes having virtually no nicotine and nitrosamines or quitting smoking altogether. Preferably, a three-step program is followed whereby at step 1, cigarettes containing 0.6 mg nicotine and less than 2 μg/g TSNA are used; at step 2, cigarettes containing 0.3 mg nicotine and less than 1 μg/g TSNA are used; and at step 3, cigarettes containing less than 0.1 mg nicotine and less than 0.7 μg/g TSNA are used. More preferably, a three-step program is followed whereby at step 1, cigarettes containing 0.6 mg nicotine and less than 2 μg/g TSNA are used; at step 2, cigarettes containing 0.3 mg nicotine and less than 1 μg/g TSNA are used; and at step 3, cigarettes containing less than 0.05 mg nicotine and less than 0.7 μg/g TSNA are used. Accordingly, the reduced nicotine and/or TSNA products described herein provide the basis for an approach to reduce the carcinogenic potential in a human in a step-wise fashion.

In a program to reduce a smoker's intake of TSNAs, (with reference to the delivery of nicotine in main-stream smoke by the FTC method) a smoker can begin the program, for example, by smoking cigarettes having 1.5 μg of nitrosamine, move to smoking cigarettes with 1 μg of nitrosamine, followed by cigarettes having 0.5 μg nitrosamine, followed by cigarettes having 0.25 μg nitrosamine, followed by cigarettes having less than 0.1 μg TSNA until the consumer decides to smoke only the cigarettes having virtually no nitrosamines or quitting smoking altogether. Preferably, a three-step program is followed whereby at step 1, cigarettes containing less than 2 μg/g TSNA are used; at step 2, cigarettes containing less than 1 μg/g TSNA are used; and at step 3, cigarettes containing less than 0.7 μg/g TSNA are used. More preferably, a three-step program is followed whereby at step 1, cigarettes containing less than 2 μg/g TSNA are used; at step 2, cigarettes containing less than 1 μg/g TSNA are used; and at step 3, cigarettes containing less than 0.7 μg/g TSNA are used. Accordingly, the reduced TSNA products described herein provide the basis for an approach to reduce the carcinogenic potential in a human in a step-wise fashion.

In some embodiments, the amount of tar in the cigarettes provided remains approximately the same from step-to-step. For example, the cigarettes provided at each step could comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) at least, less than, or equal to about 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 m, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29 mg, 29.5 mg, or 30 mg of tar. Preferably, each cigarette comprises or delivers about 8.0 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9.0 mg, 9.25 mg, or 9.5 mg of tar. It should be understood that variances between the amounts of tar in each cigarette can be acceptable in some embodiments so long as the cigarettes retain approximately the same taste characteristics as the cigarettes provided at the previous level. Preferably, cigarettes provided at each level can have variances of up to about 5 mg of tar. More preferably, cigarettes provided at each level can have variances of up to about 2.5 mg of tar. Most preferably, cigarettes provided at each level have can have variances of up to about 1.5 mg of tar.

Embodiments also include tobacco products, which are prepared with a variety of amounts of nicotine. This can be done, by blending various types of tobacco, by addition of exogenous nicotine, and/or by utilizing genetically engineered tobacco having a specific amount of nicotine. These stepwise tobacco products are made to have reduced levels of TSNAs and varying amounts of nicotine. As an example, cigarettes may comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, at least, less than or equal to 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg, 1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2.0 mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg, 2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg, 2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3.0 mg, 3.1 mg, 3.15 mg, 3.2 mg, 3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg, 3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, 4.0 mg, 4.1 mg, 4.15 mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4 mg, 4.45 mg, 4.4 mg, 4.45 mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg, 4.75 mg, 4.8 mg, 4.85 mg, 4.9 mg, 4.95 mg, or 5.0 mg of nicotine per cigarette. More preferably, blended cigarettes comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than or equal to 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg, 1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2.0 mg of nicotine. Most preferably, the cigarettes contain less than 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg of nicotine.

Another aspect of the invention is a stepwise nicotine reduction and/or tobacco-use cessation program using the reduced nicotine and/or TSNA products described above in combination with conventional NRT products. As the tobacco user progresses through the program, the amount of nicotine and/or TSNAs present in the tobacco product and/or the conventional NRT product is reduced.

As an example of a combination nicotine and/or TSNA reduction and/or tobacco-use cessation program, the program participant initially determines his or her current level of nicotine intake. The program participant then begins the program with a tobacco product having a reduced amount of nicotine, as compared to the tobacco product that was used prior to beginning the program. During the use of the low nicotine tobacco product, the user also uses an NRT (e.g., a patch, inhaler, nasal spray, lozenge, or a gum). In this respect, the tobacco cessation program provides nicotine in addition to that present in the reduced nicotine tobacco product, yet lowers the amount of inhaled nicotine—which is thought to reduce indices of nicotine dependence. Thus, the nicotine reduction and/or tobacco-use cessation program limits the exposure of a program participant to inhaled nicotine and, concomitantly, reduces the indices of nicotine dependence and retains the secondary factors of addiction, including smoke intake, oral fixation, and taste.

Such a program can utilize, for example, cigarettes that comprise (e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods), for example, at least, less than or equal to 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg, 1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2.0 mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg, 2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg, 2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3.0 mg, 3.1 mg, 3.15 mg, 3.2 mg, 3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg, 3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, 4.0 mg, 4.1 mg, 4.15 mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4 mg, 4.45 mg, 4.4 mg, 4.45 mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg, 4.75 mg, 4.8 mg, 4.85 mg, 4.9 mg, 4.95 mg, or 5.0 mg of nicotine. Such a program can utilize conventional NRT products having, for example, about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, or 25 mg of nicotine. The methods described herein facilitate tobacco-use cessation by allowing the individual to retain the secondary factors of addiction such as smoke intake, oral fixation, and taste, while gradually reducing the addictive nicotine levels consumed. Eventually, complete cessation of tobacco use is made possible because the presence of addiction for nicotine is gradually decreased while the individual is allowed to maintain dependence on the secondary factors, above. Preferred examples of a nicotine reduction and/or tobacco-use cessation program are provided in Examples 21-25.

In another aspect of the invention, the cigarettes of varying levels of nicotine are packaged to clearly indicate the level of nicotine present, and marketed as a smoking cessation program. A preferred approach to produce a product for nicotine reduction and/or tobacco-use cessation program is provided in Example 26. Individuals may wish to step up the program by skipping gradation levels of nicotine per cigarette or staying at certain steps until ready to proceed to the next level. Significantly, aspects of the invention allow a consumer to individually select the amount of nicotine that is ingested by selection of a particular tobacco product described herein. Furthermore, because the secondary factors of addiction are maintained, dependence on nicotine can be reduced rapidly.

The nicotine reduction and/or tobacco-use cessation program limits the exposure of a program participant to nicotine while retaining the secondary factors of addiction. These secondary factors include but are not limited to, smoke intake, oral fixation, and taste. Because the secondary factors are still present, the program participant may be more likely to be successful in the nicotine reduction and/or tobacco-use cessation program than in programs that rely on supplying the program participant with nicotine but remove the above-mentioned secondary factors. Ultimately, the program participant uses a tobacco product having an amount of nicotine that is less than that which is sufficient to become addictive.

In another aspect of the invention, individuals would choose to obtain only cigarettes that deliver (e.g., side-stream or main-stream smoke by the FTC and/or ISO methods) less than 0.05 mg nicotine per cigarette. Some individuals, such as individuals needing to stop nicotine intake immediately (for example, individuals with medical conditions or individuals using drugs that interact with nicotine) may find this method useful. For some individuals, the mere presence of a cigarette in the mouth can be enough to ease withdrawal from nicotine addiction. Gradually, the addictive properties of smoking can decrease since there is no nicotine in the cigarettes. These individuals are then able to quit smoking entirely.

In another aspect of the invention, packs of cigarettes containing the gradations of nicotine levels are provided as a “smoking cessation kit.” An individual who wishes to quit smoking can buy the entire kit of cigarettes at the beginning of the program. Thus any temptation that may occur while buying cigarettes at the cigarette counter is avoided. Thus, the success of this method may be more likely for some individuals. A preferred example of such a kit is provided in Example 26.

Various nicotine reduction and/or smoking cessation kits are prepared, geared to heavy, medium, or light smokers. The kits provide all of the materials needed to quit smoking in either a two-week period (fast), a one-month period (medium) or in a two-month period (slow), depending on the kit. Each kit contains a set number of packs of cigarettes modified according the present invention, containing (with reference to delivery of nicotine in the mainstream smoke by the FTC method) either step 1 cigarettes containing 0.6 mg nicotine, step 2 cigarettes containing 0.3 mg nicotine, and step 3 cigarettes containing less than 0.05 mg nicotine. For example, 1 pack a day smokers would receive 7 packs of cigarettes, each pack containing the above amounts of nicotine per each cigarette. Several weeks worth of additional cigarettes containing less than 0.05 mg nicotine/cigarette would also be provided in the kit, to familiarize the consumer with smoking no nicotine cigarettes. The kit may also contain a diary for keeping track of daily nicotine intake, motivational literature to keep the individual interested in continuing the cessation program, health information on the benefits of smoking cessation, and web site addresses to find additional anti-smoking information, such as chat groups, meetings, newsletters, recent publications, and other pertinent links.

The examples which follow are set forth to illustrate the present invention, and are not to be construed as limiting thereof.

EXAMPLE 1 Isolation and Sequencing

TobRD2 cDNA (Conkling et. al., Plant Phys. 93, 1203 (1990)) encodes QPTase, which is predicted to be a cytosolic protein. Comparisons of the NtQPT1 amino acid sequence with the GenBank database revealed limited sequence similarity to certain bacterial and other proteins; quinolate phosphoribosyl transferase (QPTase) activity has been demonstrated for the S. typhimurium, E. coli. and N. tabacum genes. The NtQPT1 encoded QPTase has similarity to the deduced peptide fragment encoded by an Arabidopsis EST (expression sequence tag) sequence (Genbank Accession number F20096), which may represent part of an Arabidopsis QPTase gene.

EXAMPLE 2 Transformation of Tobacco Plants

DNA of the QPTase gene, in antisense orientation, is operably linked to a plant promoter (CaMV 35S or TobRD2 root-cortex specific promoter) to produce two different DNA cassettes: CaMV35S promoter/antisense QPTase-encoding gene and TobRD2 promoter/antisense QPTase-encoding gene.

A wild-type tobacco line and a low-nicotine tobacco line are selected for transformation, e.g., wild-type Burley 21 tobacco (Nic1+/Nic2+) and homozygous Nic1/Nic2—Burley 21. A plurality of tobacco plant cells from each line are transformed using each of the DNA cassettes. Transformation is conducted using an Agrobacterium vector, e.g., an Agrobacterium-binary vector carrying Ti-border sequences and the nptII gene (conferring resistance to kanamycin and under the control of the nos promoter (nptII)).

Transformed cells are selected and regenerated into transgenic tobacco plants called R₀. The R₀ plants are grown to maturity and tested for levels of nicotine; a subset of the transformed tobacco plants exhibit significantly lower levels of nicotine compared to non-transformed control plants.

R₀ plants are then selfed and the segregation of the transgene is analyzed in next generation, the R₁ progeny. R₁ progeny are grown to maturity and selfed; segregation of the transgene among R₂ progeny indicates which RI plants are homozygous for the transgene.

EXAMPLE 3 Tobacco Having Reduced Nicotine and/or TSNA Levels

Tobacco of the variety Burley 21 LA was transformed with the binary Agrobacterium vector pYTY32 to produce a low nicotine tobacco variety, Vector 21-41. The binary vector pYTY32 carried the 2.0 kb NtQPT1 root-cortex-specific promoter driving antisense expression of the NtQPT1 cDNA and the nopaline synthase (nos) 3′ termination sequences from Agrobacterium tumefaciens T-DNA. The selectable marker for this construct was neomycin phosphotransferase (nptII) from E. coli Tn5 which confers resistance to kanamycin, and the expression nptII was directed by the nos promoter from Agrobacterium tumefaciens T-DNA. Transformed cells, tissues, and seedlings were selected by their ability to grow on Murashige-Skoog (MS) medium containing 300 μg/ml kanamycin. Burley 21 LA is a variety of Burley 21 with substantially reduced levels of nicotine as compared with Burley 21 (i.e., Burley 21 LA has 8% the nicotine levels of Burley 21, see Legg et al., Can J Genet Cytol, 13:287-91 (1971); Legg et al., J Hered, 60:213-17 (1969)).

One-hundred independent pYTY32 transformants of Burley 21 LA (T₀) were allowed to self. Progeny of the selfed plants (T₁) were germinated on medium containing kanamycin and the segregation of kanamycin resistance scored. T₁ progeny segregating 3:1 resulted from transformation at a single locus and were subjected to further analysis.

Nicotine levels of T₁ progeny segregating 3:1 were measured qualitatively using a micro-assay technique. Approximately ˜200 mg fresh tobacco leaves were collected and ground in 1 ml extraction solution (Extraction solution: 1 ml Acetic acid in 100 ml H₂O). Homogenate was centrifuged for 5 min at 14,000×g and supernatant removed to a clean tube, to which the following reagents were added: 100 μL NH₄OAC (5 g/100 ml H₂O+50 μL Brij 35); 500 μL Cyanogen Bromide (Sigma C-6388, 0.5 g/100 ml H₂O+50 μL Brij 35); 400 μL Aniline (0.3 ml buffered Aniline in 100 ml NH₄OAC+50 μL Brij 35). A nicotine standard stock solution of 10 mg/ml in extraction solution was prepared and diluted to create a standard series for calibration. Absorbance at 460 nm was read and nicotine content of test samples were determined using the standard calibration curve.

T₁ progeny that had less than 10% of the nicotine levels of the Burley 21 lA parent were allowed to self to produce T₂ progeny. Homozygous T₂ progeny were identified by germinating seeds on medium containing kanamycin and selecting clones in which 100% of the progeny were resistant to kanamycin (i.e., segregated 4:0; heterozygous progeny would segregate 3:1). Nicotine levels in homozygous and heterozygous T₂ progeny were qualitatively determined using the micro-assay and again showed levels less than 10% of the Burley 21 LA parent. Leaf samples of homozygous T₂ progeny were sent to the Southern Research and Testing Laboratory in Wilson, N.C. for quantitative analysis of nicotine levels using Gas Chromatography/Flame Ionization Detection (GC/FID). Homozygous T₂ progeny of transformant #41 gave the lowest nicotine levels (˜70 ppm), and this transformant was designated as “Vector 21-41.”

Vector 21-41 plants were allowed to self-cross, producing T₃ progeny. T₃ progeny were grown and nicotine levels assayed qualitatively and quantitatively. T₃ progeny were allowed to self-cross, producing T₄ progeny. Samples of the bulked seeds of the T₄ progeny were grown and nicotine levels tested.

In general, Vector 21-41 is similar to Burley 21 LA in all assessed characteristics, with the exception of alkaloid content and total reducing sugars (e.g., nicotine and nor-nicotine). Vector 21-41 may be distinguished from the parent Burley 21 LA by its substantially reduced content of nicotine, nor-nicotine and total alkaloids. As shown below, total alkaloid concentrations in Vector 21-41 are significantly reduced to approximately relative to the levels in the parent Burley 21 LA, and nicotine and nor-nicotine concentrations show dramatic reductions in Vector 21-41 as compared with Burley 21 LA. Vector 21-41 also has significantly higher levels of reducing sugars as compared with Burley 21 LA.

Field trials of Vector 21-41 T₄ progeny were performed at the Central Crops Research Station (Clayton, N.C.) and compared to the Burley 21 LA parent. The design was three treatments (Vector 21-41, a Burley 21 LA transformed line carrying only the NtQPT1 promoter [Promoter-Control], and untransformed Burley 21 LA [Wild-type]), 15 replicates, 10 plants per replicate. The following agronomic traits were measured and compared: days from transplant to flowering; height at flowering; leaf number at flowering; yield; percent nicotine; percent nor-nicotine; percent total nitrogen; and percent reducing sugars.

Vector 21-41 was also grown on approximately 5000 acres by greater than 600 farmers in five states (Pennsylvania, Mississippi, Louisiana, Iowa, and Illinois). The US Department of Agriculture, Agriculture Marketing Service (USDA-AMS) quantified nicotine levels (expressed as percent nicotine per dry weight) using the FTC method of 2,701 samples taken from these farms. Nicotine levels ranged from 0.01% to 0.57%. The average percent nicotine level for all these samples was 0.09%, with the median of 0.07%. Burley tobacco cultivars typically have nicotine levels between 2% and 4% dry weight (Tso, T. C., 1972, Physiology and Biochemistry of Tobacco Plants. Dowden, Hutchinson, and Ross, Inc. Stroudsbury).

EXAMPLE 4 Regulation of NtQPT1 Gene Expression Using Molecular Decoys

Nucleotide sequence located between −1000 and −600 or −700 bp of the NtQPT1 promoter is inserted in tandem arrays into a plant—Agrobacterium shuttle vector and subsequently transformed into tobacco via methods known to one skilled in the art. Plants stably transformed with said vector are assessed for the level of expression of NtQPT1 and for nicotine and/or TSNA content. These experiments demonstrate that tobacco transformed with molecular decoys that interact with Nic gene products exhibit a reduced level of expression of NtQPT1.

EXAMPLE 5 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using Molecular Decoys

Multiple copies of an approximately 300 or 400 nucleotide long fragment of the NtQPT1 promoter (e.g., including nucleotide sequence located between −1000 and −600 or −700 bp of the NtQPT1 promoter), are affixed to microparticles (e.g., by precipitation) that are suitable for the ballistic transformation of a plant cell (e.g., 1 to 5 μm gold spheres). The microparticles are propelled into tobacco plant cells (e.g., Burley 21 LA) using any suitable ballistic cell transformation methodology, so as to produce transformed plant cells. Plants are then regenerated from the transformed plant cells. Burley 21 LA is a variety of Burley 21 with substantially reduced levels of nicotine as compared with Burley 21 (i.e., Burley 21 LA has 8% the nicotine levels of Burley 21, see Legg et al., Can J Genet Cytol, 13:287-91 (1971); Legg et al., J Hered, 60:213-17 (1969))

Transformed cells, tissues, and seedlings are grown on Murashige-Skoog (MS) medium (with or without the selection compound, e.g., antibiotic, depending on whether a selectable marker was used. One-hundred independent transformants of Burley 21 LA (T₀) are allowed to self. Progeny of the selfed plants (T₁) are germinated. Nicotine levels of T₁ progeny are measured qualitatively using a micro-assay technique. Approximately 200 mg fresh tobacco leaves are collected and ground in 1 ml extraction solution. (Extraction solution: 1 ml Acetic acid in 100 ml H₂O) Homogenate is centrifuged for 5 min at 14,000×g and supernatant removed to a clean tube, to which the following reagents are added: 100 μL NH₄OAC (5 g/100 ml H₂O+50 μl, Brij 35); 500 μL Cyanogen Bromide (Sigma C-6388, 0.5 g/100 ml H₂O+50 μL Brij 35); 400 μL Aniline (0.3 ml buffered Aniline in 100 ml NH₄OAC+50 μL Brij 35). A nicotine standard stock solution of 10 mg/ml in extraction solution is prepared and diluted to create a standard series for calibration. Absorbance at 460 nm is read and nicotine content of test samples are determined using the standard calibration curve.

T₁ progeny that have less than 10% of the nicotine levels of the Burley 21 LA parent are allowed to self to produce T₂ progeny. Homozygous T₂ progeny are then identified. Nicotine levels in homozygous and heterozygous T₂ progeny are also qualitatively determined using the micro-assay. Leaf samples of homozygous T₂ progeny can also be sent to the Southern Research and Testing Laboratory in Wilson, N.C. for quantitative analysis of nicotine levels using Gas Chromatography/Flame Ionization Detection (GC/FID). Homozygous T₂ progeny will have nicotine levels that are substantially reduced as compared to the untransformed tobacco (e.g., ˜70 ppm). Because the nicotine levels in such plants are substantially reduced, the TSNA levels in these plants are concomitantly reduced.

These experiments demonstrate that tobacco transformed with molecular decoys that interact with Nic gene products exhibit a reduced amount of nicotine and/or TSNA. Plants with multiple tandem insertions of the molecular decoy that have reduced NtQPT1 expression and reduced nicotine/TSNA levels are used to generate commercially valuable tobacco products.

EXAMPLE 6 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

A transgenic Flue-cured tobacco with a reduced amount of nicotine and TSNAs was created using an RNAi approach. FIG. 1 illustrates an RNAi construct that was used to create a reduced nicotine tobacco, wherein the root-specific promoter RD2 (Bp 1-2010) was used to drive expression of an RNAi cassette comprising an antisense full-length QPTase cDNA (Bp 2011-3409) linked to a 382 bp fragment of the cucumber aquaporin gene (Bp 3410-3792), which is linked to a sense full-length QPTase cDNA (Bp 3793-5191) and the GapC terminator (Bp5192-5688) (see SEQ. ID. No. 21). This first RNAi construct also comprises a GUS-selection cassette comprising the GapC promoter (Bp 1-1291), which drives expression of the GUS gene (Bp 1292-3103), linked to the GapC terminator (Bp 3104-3600) (see SEQ. ID. No. 24). This first RNAi construct was ligated into a binary vector, pBin19 which was then introduced into Agrobacterium tumefaciens. Leaf disks from flue-cured variety K326 were then transformed with Agrobacterium that contained the RNAi construct comprising the RNAi cassette and the GUS selection cassette. GUS-based selection was then employed to select positively transformed plantlets (buds), which were then regenerated to plants. Leaf samples were then harvested and the alkaloid content was then determined. The alkaloid content of samples obtained from some of the transgenic lines created with this first RNAi construct was 6000 ppm. Since the total alkaloid content in tobacco is about 90% nicotine, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 1 has reduced levels of nicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 7 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

FIG. 2 shows another RNAi construct that was used to generate several lines of reduced nicotine and TSNA tobacco. This RNAi construct has a QPTase inhibition cassette (SEQ. ID. No. 22) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the QPTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense fragment (360 bp) (Bp 2011-2370) of the QPTase gene, joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense fragment of the QPTase gene (360 bp) (Bp 3502-3861), which is joined to the GAD2 terminator (Bp 3862-4134). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSMI) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco was transformed with the construct shown in FIG. 2 using Agrobacterium-mediated transformation and 1,140 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 1,140 independent lines, 1,097 plants were harvested and tested for alkaloid content. A total of 608 lines were identified as having less than 1,000 ppm total alkaloid and 139 lines were identified as having less than 500 ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco created using the construct shown in FIG. 2 has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 8 Tobacco Heaving Reduced Nicotine and/or TSNA Levels Generated Using RNAi

Burley tobacco was also transformed with the construct shown in FIG. 2 using Agrobacterium-mediated transformation and 385 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 385 independent lines, 350 lines of plants were harvested and tested for alkaloid content. A total of 142 lines were identified as having less than 1,000 ppm total alkaloid and 10 lines were identified as having less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Burley tobacco created using the construct shown in FIG. 2 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 9 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

Oriental tobacco was transformed with the construct shown in FIG. 2 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and 61 independent lines were selected, regenerated, and transplanted in the greenhouse. All 61 lines were tested for alkaloid content and a total of 10 lines were identified as having less than 1,500 ppm total alkaloids and a total of 3 lines were identified as having less than 1,000 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Oriental tobacco created using the construct shown in FIG. 2 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 10 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

FIG. 3 illustrates another RNAi construct that can be used to create a reduced nicotine and TSNA transgenic tobacco. This RNAi construct has a PMTase inhibition cassette (SEQ. ID. No. 5) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the PMTase inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic acid (241 bp) (Bp 2011-2251) of a PMTase gene, joined to a FAD2 intron (Bp 2252-3382), which is joined to a sense nucleic acid of the PMTase gene (241 bp) (Bp 3383-3623), which is joined to the GAD2 terminator (Bp 3624-3896). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco will be transformed with the construct shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Flue-cured tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 11 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

Burley tobacco will be transformed with the construct shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated (see e.g., Broothaerts et al., Nature 433:629 (2005), herein expressly incorporated by reference in its entirety) or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 12 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

Oriental tobacco will also be transformed with the construct shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 3 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 13 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

FIG. 4 illustrates another RNAi construct that was used to create a reduced nicotine and TSNA transgenic tobacco. This RNAi construct has a A622 inhibition cassette (SEQ. ID. No. 3) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the A622 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic acid (628 bp) (Bp 2011-2638) of the A622 gene, joined to a FAD2 intron (Bp 2639-3769), which is joined to a sense nucleic acid of the A622 gene (628 bp) (Bp 3770-4397), which is joined to the GAD2 terminator (Bp 4398-4670). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco was transformed with the construct shown in FIG. 4 using Agrobacterium-mediated transformation and 270 independent lines were selected, regenerated, and transplanted in the greenhouse. Of the 270 independent lines, 259 plants were harvested and tested for alkaloid content. A total of 131 lines were identified as having less than 1,000 ppm total alkaloid and 45 lines were identified as having less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 4 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

Several lines that were transformed with this construct were unexpectedly found to have conventional levels of nicotine but a significantly reduced amount of nornicotine. That is, 9 lines were found to have nicotine levels ranging from 2.17 mg/g to 3.99 mg/g and nornicotine levels less than or equal to 0.00 to 0.06 mg/g (see Table 2). TABLE 2 Transgenic tobacco having reduced nornicotine and conventional amounts of nicotine Alkaloid Nornicotine Nicotine new I.D (ppm) (mg/g) (mg/g) VDG 020 2486.53

2.30 VDG 032 4683.01

3.48 VDG 045 4490.79

3.94 VDG 052 2855.58

2.61 VDG 054 2291.89

2.17 VDG 077 4857.86

3.99 VDG 097 3072.40

2.58 VDG 107 4921.31

3.59 VDG 116 4960.64

3.56 Control-8 5005.22 0.28 4.02 Control-20 5711.97 0.34 5.35 Control-28 5196.25 0.24 4.52 *Highlighted entries show transgenic tobacco lines having a reduced amount of nornicotine and conventional amounts of nicotine. *Highlighted entries show transgenic tobacco lines having a reduced amount of nornicotine and conventional amounts of nicotine.

EXAMPLE 14 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

Burley tobacco will be transformed with the construct shown in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 4 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification. It is also expected that some lines of tobacco created with the afore-mentioned nucleic acid construct will retain conventional amounts of nicotine but will comprise a reduced amount of nornicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 15 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using RNAi

Oriental tobacco will also be transformed with the construct shown in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 4 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification. It is also expected that some lines of tobacco created with the afore-mentioned nucleic acid construct will retain conventional amounts of nicotine but will comprise a reduced amount of nornicotine, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 16 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using a Double Knock-Out RNAi Construct

FIG. 5 illustrates a double-knock-out RNAi construct, which has been created to develop a reduced nicotine and TSNA transgenic tobacco. This double-knock-out RNAi construct has a QPTase/A622 inhibition cassette (SEQ. ID. No.23) and a norflurazone selection cassette (SEQ. ID. No. 25). Starting from the right border (RB), the QPTase/A622 inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably linked to a QPTase antisense nucleic acid (360 bp) (Bp 2011-2370) of a QPTase gene, which is joined to a A622 antisense nucleic acid (628 bp) (Bp 2371-2998) of a A622 gene, which is joined to a FAD2 intron (Bp 2999-4129), which is joined to a sense nucleic acid of the A622 gene (628bp) (Bp 4130-4757), which is joined to a sense nucleic acid of the QPTase gene (360 bp) (Bp 4758-5117), which is joined to the GAD2 terminator (Bp 5118-5390). The selection cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387) at the left border (LB).

Flue-cured tobacco was transformed with the construct shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated, or biolistic transformation and 444 independent lines were selected, regenerated, and transplanted in the greenhouse. Each of these lines were analyzed for alkaloid content and 240 lines were found to have less than 1,000 ppm total alkaloid and 18 lines were found to have less than 500 ppm total alkaloid. Accordingly, it is understood by those skilled in the art that the transgenic Flue-cured tobacco created using the construct shown in FIG. 5 also has significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 17 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using a Double Knock-Out RNAi Construct

Burley tobacco will be transformed with the construct shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Burley tobacco that will be created using the construct shown in FIG. 5 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 18 Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using a Double Knock-Out RNAi Construct

Oriental tobacco will also be transformed with the construct shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated, or biolistic transformation and independent lines will be selected, regenerated, and transplanted in the greenhouse. Most of the independent lines grown in the greenhouse will be harvested and tested for alkaloid content. It is expected that approximately 50% of the lines tested will have less than 1,000 ppm total alkaloid and approximately 10% of the lines tested will have less than 500 ppm total alkaloid. Accordingly, it is expected that the transgenic Oriental tobacco that will be created using the construct shown in FIG. 5 will have significantly reduced levels of nicotine and TSNA, as compared to a conventional tobacco, a reference tobacco, or the parental strain of tobacco prior to genetic modification.

EXAMPLE 19 Low Nicotine and Nitrosamine Blended Tobacco

The following example describes several ways to create tobacco products having specific amounts of nicotine and/or TSNAs through blending. Some blending approaches begin with tobacco prepared from varieties that have extremely low amounts of nicotine and/or TSNAs. With reference to nicotine and/or TSNA delivery in mainstream smoke by the FTC method, by blending prepared tobacco from a low nicotine/TSNA variety (e.g., undetectable levels of nicotine and/or TSNAs ) with a conventional tobacco (e.g., Burley, which has 30,000 parts per million (ppm) nicotine and 8,000 parts per billion (ppb) TSNA; Flue-Cured, which has 20,000 ppm nicotine and 300 ppb TSNA; and Oriental, which has 10,000 ppm nicotine and 100 ppb TSNA), tobacco products having virtually any desired amount of nicotine and/or TSNAs can be manufactured. Other approaches blend only low nicotine/TSNA tobaccos (e.g., genetically modified Burley, genetically modified Virginia flue, genetically modified Oriental tobaccos that contain reduced amounts of nicotine and/or TSNAs, and tobacco that has been treated to remove nicotine). Tobacco products having various amounts of nicotine and/or TSNAs can be incorporated into tobacco-use cessation kits and programs to help tobacco users reduce or eliminate their dependence on nicotine and reduce the carcinogenic potential.

By one approach, a step 1 tobacco product is comprised of approximately 25% low nicotine/TSNA tobacco and 75% conventional tobacco; a step 2 tobacco product can be comprised of approximately 50% low nicotine/TSNA tobacco and 50% conventional tobacco; a step 3 tobacco product can be comprised of approximately 75% low nicotine/TSNA tobacco and 25% conventional tobacco; and a step 4 tobacco product can be comprised of approximately 100% low nicotine/TSNA tobacco and 0% conventional tobacco. A tobacco-use cessation kit can comprise an amount of tobacco product from each of the aforementioned blends to satisfy a consumer for a single month program. That is, if the consumer is a one pack per day smoker, for example, a single month kit would provide 7 packs from each step, a total of 28 packs of cigarettes. Each tobacco-use cessation kit would include a set of instructions that specifically guide the consumer through the step-by-step process. Of course, tobacco products having specific amounts of nicotine and/or TSNAs would be made available in conveniently sized amounts (e.g., boxes of cigars, packs of cigarettes, tins of snuff, and pouches or twists of chew) so that consumers could select the amount of nicotine and/or TSNA they individually desire. There are many ways to obtain various low nicotine/low TSNA tobacco blends using the teachings described herein and the following is intended merely to guide one of skill in the art to one possible approach.

To obtain a step 1 tobacco product, which is a 25% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 25%/75% ratio respectively to obtain a Burley tobacco product having 22,500 ppm nicotine and 6,000 ppb TSNA, a Flue-cured product having 15,000 ppm nicotine and 225 ppb TSNA, and an Oriental product having 7,500 ppm nicotine and 75 ppb TSNA. Similarly, to obtain a step 2 product, which is 50% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 50%/50% ratio respectively to obtain a Burley tobacco product having 15,000 ppm nicotine and 4,000 ppb TSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb TSNA, and an Oriental product having 5000 ppm nicotine and 50 ppb TSNA. Further, a step 3 product, which is a 75%/25% low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a 75%/25% ratio respectively to obtain a Burley tobacco product having 7,500 ppm nicotine and 2,000 ppb TSNA, a Flue-cured product having 5,000 ppm nicotine and 75 ppb TSNA, and an Oriental product having 2,500 ppm nicotine and 25 ppb TSNA.

It should be appreciated that tobacco products are often a blend of many different types of tobaccos, which were grown in many different parts of the world under various growing conditions. As a result, the amount of nicotine and TSNAs will differ from crop to crop. Nevertheless, by using conventional techniques one can easily determine an average amount of nicotine and TSNA per crop used to create a desired blend. By adjusting the amount of each type of tobacco that makes up the blend one of skill can balance the amount of nicotine and/or TSNA with other considerations such as appearance, flavor, and smokability. In this manner, a variety of types of tobacco products having varying level of nicotine and/or nitrosamine, as well as, appearance, flavor and smokability can be created.

EXAMPLE 20 Low Nicotine and TSNA Blended Tobacco

By a preferred method, conventional Virginia flue tobacco was blended with genetically modified Burley (i.e., Burley containing a significantly reduced amount of nicotine and nitrosamine) to yield a blended tobacco that was incorporated into three levels of reduced nicotine cigarettes (Quest 1®, Quest2®, and Quest3®): a step 1 cigarette containing 0.6 mg nicotine, a step 2 cigarette containing 0.3 mg nicotine, and a step 3 cigarette containing less than 0.05 mg nicotine (nicotine delivery in mainstream smoke by the FTC method). The amount of total TSNA was found to range between approximately 0.17 μg/g-0.6 μg/g (TSNA delivery in mainstream smoke by the FTC method).

In some cigarettes, approximately, 28% of the blend was Virginia flue tobacco approximately 29% of the blend was genetically modified (i.e., reduced nicotine Burley), approximately 14% of the blend was Oriental, approximately 17% of the blend was expanded flue-cured stem, and approximately 12% was standard commercial reconstituted tobacco. The amount of total TSNAs in cigarettes containing this blend was approximately 1.5 μg/g (TSNA delivery in mainstream smoke by the FTC method).

TABLES 3, 4, and 5 show the results of a study that analyzed the amount of nicotine present in the blended tobacco used to make the Quest 1® and Quest2® cigarettes, as compared to the Vector 21-41 tobacco used to generate the Quest3® tobacco product, which is not a blended cigarette. TABLE 3 QUEST 1 Average mg/g Lot#031805- BLEND Nicotine per dry 1H9R Sample ID weight basis Range % CV Quest 1 (1) 14.291 0.230 0.48 Quest 1 (2) 14.958 0.230 1.54 Quest 1 (3) 15.207 0.021 0.14 Quest 3 “control” 0.533 0.007 1.28 Q1 AVG 14.818 STD 0.474 % CV 0.032

Analysis done according to 1999 National Registry Method.

Nicotine reported in a dry weight basis TABLE 4 GC ANAL. DATE Lot#031 Nicotine 105- Content CLINIC 2F7P after QUEST 2 TRIAL Nicotine corrected BLEND Weight Content for % mg/g Avg. Sample of Tob. from GC % Moisture of mg/g ID (g) (mg) Moist. (mg) Tob. Nic. Range % CV 1 A 1.002 8.136 8.230 8.213 B 1.000 8.173 1.14 8.267 8.267 8.240 0.038 0.47 2 A 1.001 7.679 7.810 7.802 B 1.003 7.662 1.67 7.792 7.768 7.785 0.017 0.22 3 A 1.001 6.666 6.809 6.802 B 1.000 7.020 2.1  7.171 7.171 6.986 0.261 3.73 Q3 A 1.002 0.488 0.497 0.496 control B 1.001 0.501 1.89 0.510 0.510 0.503 0.010 1.93

TABLE 5 Average mg/g QUEST 3 Nicotine per dry Sample ID weight basis Range % CV 1 0.902 0.006 0.63 2 0.905 0.010 1.14 3 0.885 0.008 0.88 Q3 control 0.503 0.010 1.93 AVG 0.897 STD 0.011 % CV 1.20%

Analysis done according to 1999 National Registry Method.

Nicotine reported in a dry weight basis

TABLE 6 shows the amounts of nicotine present in the mainstream smoke generated by Quest 1®, Quest2®, and Quest3® tobacco products (as determined by the FTC method), as compared to two different commercially sold cigarettes (cigarette A, a “100” type and cigarette B, a “light” type) and a reference cigarette (2R4F). The data shows that the Quest 1®, Quest2®, and Quest3® products deliver significantly less nicotine than the two commercially available cigarettes and the reference cigarette. It should be noted that the Quest 3® analyzed in this study is not a blended tobacco in that it only contains Vector 21-41 tobacco. TABLE 6 Client Arista MS TPM CO Water Nicotine Tar Run# Port# Code Code Puffs/Cigt (mg/cigt) (mg/cigt) (mg/cigt) (mg/cigt) (mg/cigt) C00420 E Cig A 3557 11.3 24.74 16.66 3.96 1.48 19.30 C00420 J Cig A 3557 11.3 23.58 16.31 2.49 1.52 19.58 C00420 Q Cig A 3557 11.6 24.66 18.05 3.56 1.53 19.57 C00421 J Cig A 3557 11.2 24.14 17.36 2.79 1.49 19.86 C00421 O Cig A 3557 11.5 23.66 16.82 2.74 1.49 19.43 C00421 Q Cig A 3557 11.0 23.96 18.04 2.32 1.47 20.17 Average 11.3 24.12 17.21 2.98 1.50 19.65 sd 0.2 0.49 0.73 0.64 0.02 0.32 % RSD 1.9 2.0 4.3 21.7 1.4 1.6 C00420 C Cig B 3558 8.6 12.72 11.60 1.09 0.83 10.79 C00420 I Cig B 3558 8.1 12.18 12.69 0.86 0.81 10.50 C00420 K Cig B 3558 8.1 12.56 10.90 0.99 0.81 10.76 C00420 M Cig B 3558 8.1 12.46 10.93 1.07 0.80 10.59 C00420 O Cig B 3558 8.1 12.20 10.96 0.94 0.81 10.44 C00421 R Cig B 3558 8.6 13.50 11.70 0.95 0.86 11.70 Average 8.3 12.60 11.46 0.98 0.82 10.80 sd 0.3 0.49 0.70 0.09 0.02 0.46 % RSD 3.4 3.9 6.1 8.8 2.5 4.3 C00421 A Quest 1 3559 7.1 11.12 11.46 1.09 0.48 9.54 C00421 D Quest 1 3559 6.6 11.22 11.36 0.93 0.49 9.80 C00421 F Quest 1 3559 6.6 10.98 10.68 1.08 0.47 9.44 C00421 H Quest 1 3559 6.3 10.78 10.44 1.12 0.47 9.20 C00421 K Quest 1 3559 6.8 11.20 11.28 1.18 0.45 9.57 C00421 M Quest 1 3559 6.7 11.04 11.19 0.65 0.48 9.91 Average 6.7 11.06 11.07 1.01 0.47 9.58 sd 0.3 0.16 0.41 0.19 0.01 0.26 % RSD 3.8 1.5 3.7 19.1 3.0 2.7 C00420 D Quest 2 3560 6.4 10.88 11.89 0.70 0.29 9.90 C00420 S Quest 2 3560 6.6 10.52 12.03 1.03 0.27 9.21 C00421 B Quest 2 3560 6.7 12.18 11.91 1.67 0.31 10.20 C00421 I Quest 2 3560 6.2 10.44 10.88 0.97 0.25 9.21 C00421 L Quest 2 3560 6.2 10.78 11.25 1.32 0.28 9.18 Average 6.4 10.96 11.59 1.14 0.28 9.54 sd 0.2 0.71 0.50 0.37 0.02 0.48 % RSD 3.9 6.4 4.3 32.7 7.0 5.0 C00420 B Quest 3 3561 6.1 8.72 9.79 0.79 0.049 7.89 C00420 P Quest 3 3561 6.1 7.94 9.76 0.44 0.047 7.46 C00420 R Quest 3 3561 6.3 8.58 10.39 0.58 0.052 7.95 C00421 C Quest 3 3561 6.2 9.22 10.30 0.89 0.045 8.28 C00421 N Quest 3 3561 6.2 8.62 10.38 0.95 0.047 7.62 Average 6.2 8.62 10.12 0.73 0.048 7.84 sd 0.1 0.46 0.32 0.22 0.003 0.32 % RSD 1.5 5.3 3.2 29.6 5.7 4.1 C00420 N 2R4F 912 9.0 10.80 11.50 0.62 0.79 9.39 C00421 G 2R4F 912 9.1 12.24 13.32 0.92 0.83 10.49 C00421 P 2R4F 912 9.3 11.50 12.09 0.78 0.80 9.92 Average 9.1 11.51 12.30 0.77 0.81 9.93 sd 0.2 0.72 0.93 0.15 0.02 0.55 % RSD 1.8 6.3 7.5 19.4 2.9 5.5

TABLE 7 shows the amounts of TSNAs present in mainstream smoke generated by Quest 1®, Quest2®, and Quest3® (as determined by the FTC method), as compared to two different commercially sold cigarettes (cigarette A, a “100” type and cigarette B, a “light” type) and a reference cigarette (2R4F). The data shows that the Quest 1®, Quest2®, and Quest3® products deliver significantly less TSNAs than the two commercially available cigarettes and the reference cigarette. It should be noted that the Quest 3® analyzed in this study is not a blended tobacco in that it only contains Vector 21-41 tobacco. TABLE 7 Client Arista Puffs/ MS TPM NAB NAT NNK NNN Run# Port Code Code Cigt (mg/cigt) (ng/cigt) (ng/cigt) (ng/cigt) (ng/cigt) C004 B CIG 3557 11.5 24.2 25.8 203 103 235 15 A C004 P CIG 3557 11.0 22.4 27.6 200 114 197 15 A C004 R CIG 3557 11.0 20.8 25.0 186 103 207 15 A C004 T CIG 3557 11.3 22.9 26.7 213 111 216 15 A C004 I CIG 3557 11.3 22.9 26.1 200 106 204 18 A C004 O CIG 3557 11.4 23.9 23.3 201 112 223 18 A Average 11.3 22.9 25.8 201 108 214 SD 0.2 1.2 1.5 9 5 14 % RSD 1.8 5.3 5.8 4.3 4.6 6.5 C004 E CIG 3558 8.4 12.9 16.8 127 75.5 138 15 B C004 I CIG 3558 8.2 13.4 17.6 135 89.6 144 15 B C004 K CIG 3558 7.8 12.6 16.0 115 79.9 144 15 B C004 S CIG 3558 8.3 12.6 18.9 138 84.5 138 15 B C004 J CIG 3558 8.1 12.6 17.1 135 83.5 128 18 B C004 M CIG 3558 7.8 12.0 14.0 119 77.3 138 18 B Average 8.1 12.7 16.7 128 81.7 138 SD 0.3 0.4 1.7 10 5.2 6 % RSD 3.3 3.5 9.9 7.5 6.4 4.3 C004 C Quest 3559 6.7 12.5 6.59 41.5 27.3 62.7 15 1 C004 L Quest 3559 6.6 10.6 6.20 40.6 25.6 64.9 15 1 C004 N Quest 3559 6.0 9.84 6.39 37.9 26.3 56.1 15 1 C004 F Quest 3559 7.0 11.4 6.27 37.7 27.6 56.5 18 1 C004 H Quest 3559 6.6 10.3 5.39 39.1 24.6 51.3 18 1 C004 L Quest 3559 6.6 10.0 5.90 41.9 26.5 58.4 18 1 Average 6.6 10.8 6.13 39.8 26.3 58.3 SD 0.3 1.0 0.43 1.8 1.1 4.9 % RSD 4.8 9.2 7.0 4.6 4.2 8.4 C004 D Quest 3560 6.3 9.72 3.64 26.3 29.5 62.2 15 2 C004 G Quest 3560 6.0 9.80 3.34 23.8 29.6 52.4 15 2 C004 J Quest 3560 6.2 10.4 3.67 23.5 31.3 59.4 15 2 C004 N Quest 3560 6.1 10.6 3.72 23.4 31.4 58.3 18 2 C004 P Quest 3560 6.3 10.5 3.53 22.4 31.3 56.1 18 2 C004 R Quest 3560 6.4 10.9 3.91 21.5 38.2 54.1 18 2 Average 6.2 10.3 3.63 23.5 31.9 57.1 SD 0.1 0.5 0.19 1.6 3.2 3.6 % RSD 2.1 4.5 5.3 7.0 10.1 6.3 C004 F Quest 3561 6.0 8.64 1.1 5.34 10.4 78.4 15 3 C004 H Quest 3561 6.0 8.46 1.0 6.13 13.2 79.0 15 3 C004 O Quest 3561 6.1 8.68 1.1 6.63 14.6 76.0 15 3 C004 C Quest 3561 6.2 9.76 1.2 6.66 11.6 78.9 18 3 C004 E Quest 3561 6.2 8.88 1.0 6.31 14.5 81.1 18 3 C004 K Quest 3561 6.0 8.94

6.42 16.0 81.9 18 3 Average 6.1 8.89

6.25 13.4 79.2 SD 0.1 0.46 0.1 0.49 2.1 2.1 % RSD 1.3 5.2 7.4 7.8 15.6 2.7 C004 M 2R4F  912 9.0 11.1 17.2 124 137 145 15 C004 Q 2R4F  912 9.1 10.8 17.2 141 146 147 15 C004 D 2R4F  912 9.2 11.8 16.5 126 143 153 18 C004 G 2R4F  912 9.0 11.9 17.2 137 150 146 18 Average 9.1 11.4 17.0 132 144 148 SD 0.1 0.5 0.4 8 5 4 % RSD 1.0 4.8 2.2 6.1 3.8 2.5 LOD: 0.8 1.4 0.4 1.0 LOQ: 2.5 4.5 1.4 3.5 Note Shaded cells contain values between the Limit of Detection (LOD) and Limit of Quantitation (LOQ) for the method.

In one study, a Canadian intense smoking regimen was performed, wherein it was demonstrated that Quest 3® delivers 0.05 mg/cig using extreme smoking parameters. (See TABLES 8 and 9). Note, that Quest 1® delivers 1.07 mg/cig under these conditions as opposed to 0.60 using FTC smoking conditions. Similar results are observed for Quest 2®. TABLE 8 Puff Count Nicotine Tar Matrix Condition Sample (/cig) (mg/cig) (mg/cig) Code Code ID Average St Dev Average St Dev Average St Dev MS N 040501 8.6 0.2 1.07 0.06 22.1 1.4 MS N 040502 8.6 0.2 0.606 0.030 21.8 1.4 MS N 040503 8.0 0.3 0.050 0.003 19.8 1.2

Glossary of Abbreviations

Condition Code:

N—puff volume, 55 mL; interval, 30 sec; duration, 2 sec; vent blocking, 100%. Brand Sample ID Description 040501 Quest ® Lights 1 Low Nicotine 040502 Quest ® Lights 2 Extra Low Nicotine 040503 Quest ® Lights 3 Nicotine Free

TABLE 9 Yields of “Tar” and Nicotine in Mainstream Tobacco Smoke: ‘Intense’ Conditions* Sample Weight Puff Count MS TPM CO Water Nicotine Tar ID (mg/cig) (per cig) (mg/cig) (mg/cig) (mg/cig) (mg/cig) (mg/cig) 040501 1044 8.1 32.4 10.2 1.04 21.2 040501 1043 8.4 35.2 11.4 1.05 22.7 040501 1062 8.6 35.4 11.7 1.08 22.6 040501 1057 8.7 33.0 11.1 1.04 20.9 040501 1059 8.7 35.4 12.5 1.07 21.8 040501 1061 8.8 35.4 11.2 1.12 23.0 040501 1067 8.7 31.6 9.70 1.04 20.8 040501 1054 8.4 35.0 10.9 1.09 23.0 040501 1060 8.7 32.3 9.58 1.07 21.7 040501 1055 8.5 36.1 12.3 1.01 22.8 040501 1058 8.8 38.4 12.6 1.16 24.6 040501 1042 8.3 31.7 9.92 0.977 20.8 040501 1042 9.0 36.4 11.7 1.18 23.5 040501 1061 8.9 32.9 10.5 1.07 21.3 040501 1054 8.3 32.4 10.5 1.03 20.9 040501 1064 8.6 32.4 10.7 1.02 20.7 040501 1068 8.4 32.7 10.8 1.04 20.8 040501 1051 8.4 31.0 10.1 0.995 20.0 040501 1037 8.3 38.1 11.7 1.13 25.2 040501 1058 8.6 35.1 10.7 1.16 23.3 Average 1055 8.6 34.1 11.0 1.07 22.1 Std. Dev. 9 0.2 2.2 0.9 0.06 1.4 Coeff. Var. 0.9 2.7 6.4 8.2 5.4 6.5 040502 1066 8.6 32.7 10.9 0.642 21.2 040502 1067 8.7 36.5 12.2 0.659 23.7 040502 1062 8.6 29.1 9.22 0.576 19.3 040502 1039 8.7 32.5 10.5 0.599 21.3 040502 1058 8.5 34.8 11.5 0.572 22.7 040502 1049 8.9 36.0 12.0 0.608 23.5 040502 1051 8.8 31.4 10.3 0.608 20.5 040502 1049 8.2 34.2 11.4 0.585 22.2 040502 1053 8.5 35.6 12.0 0.639 23.0 040502 1073 8.8 34.8 11.0 0.633 23.2 040502 1054 8.2 29.6 9.43 0.529 19.6 040502 1062 8.6 32.9 10.3 0.577 22.0 040502 1068 8.6 35.2 11.4 0.608 23.1 040502 1061 8.9 35.9 11.8 0.633 23.5 040502 1089 8.8 35.4 12.1 0.618 22.6 040502 1084 8.6 34.2 11.6 0.593 22.0 040502 1028 8.6 33.3 11.8 0.586 20.9 040502 1097 9.0 32.5 11.5 0.617 20.4 040502 1077 8.7 30.5 9.65 0.625 20.3 040502 1060 8.4 31.3 9.46 0.616 21.3 Average 1062 8.6 33.4 11.0 0.606 21.8 Std. Dev. 17 0.2 2.2 1.0 0.030 1.4 Coeff. Var. 1.6 2.5 6.6 8.9 5.0 6.2 040503 1005 8.0 29.9 9.95 0.046 19.9 040503 996 7.4 26.7 8.50 0.045 18.1 040503 1029 8.4 30.4 10.3 0.054 20.1 040503 1015 7.9 31.9 10.7 0.051 21.2 040503 1026 8.2 30.8 10.0 0.050 20.7 040503 1011 8.3 28.2 8.91 0.049 19.2 040503 1014 8.4 29.7 9.26 0.052 20.4 040503 1013 8.5 29.6 9.57 0.050 19.9 040503 1002 7.7 29.5 9.86 0.046 19.6 040503 1007 8.3 31.0 10.6 0.053 20.4 040503 1012 8.0 30.0 10.3 0.047 19.6 040503 1000 7.8 32.8 12.1 0.050 20.7 040503 1005 7.7 32.4 11.7 0.046 20.6 040503 1023 8.1 28.3 9.57 0.048 18.7 040503 1028 8.5 25.5 8.39 0.047 17.1 040503 1010 8.3 31.5 10.5 0.051 20.9 040503 1009 7.9 32.2 11.8 0.054 20.4 040503 1011 8.1 30.0 11.2 0.048 18.8 040503 1016 7.7 31.7 10.0 0.058 21.6 040503 994 7.3 25.4 7.47 0.053 17.9 Average 1011 8.0 29.9 10.0 0.050 19.8 Std. Dev. 10 0.3 2.1 1.2 0.003 1.2 Coeff. Var. 1.0 4.2 7.2 11.7 6.8 6.0 *puff volume, 55 mL; interval, 30 sec; duration, 2 sec; vent blocking, 100%. See text for additional details.

EXAMPLE 21 Nicotine Reduction and/or Smoking Cessation Program Utilizing Reduced Nicotine Tobacco Products

The following example describes a nicotine reduction and/or smoking cessation program utilizing the low nicotine, low TSNA tobacco products of the present invention. The modified tobacco containing very low levels of TSNAs and essentially no nicotine was mixed with tobacco having a known amount of nicotine to create specific, stepwise levels of nicotine per cigarette. As an example, Virginia flue tobacco was blended with genetically modified Burley (i.e., Burley containing a significantly reduced amount of nicotine and nitrosamine) to yield a blended tobacco that was incorporated into three levels of reduced nicotine cigarettes (with reference to nicotine and/or TSNA delivery in mainstream smoke by the FTC method): a step 1 cigarette containing 0.6 mg nicotine, a step 2 cigarette containing 0.3 mg nicotine, and a step 3 cigarette containing less than 0.05 mg nicotine. The stepwise packs of cigarettes were clearly marked as to their nicotine content, and the step in the stepwise nicotine reduction program was also clearly marked on the package. Each week, the user purchases packs containing cigarettes having the next lower level of nicotine, but limits himself to no more cigarettes per day than consumed previously. The user may define his/her own rate of nicotine reduction and/or smoking cessation according to individual needs by choosing a) the number of cigarettes smoked per day b) the starting nicotine levels c) the change in nicotine level per cigarette each week, and d) the final level of nicotine consumed per day. To keep better track of the program, the individual keeps a daily record of total nicotine intake, as well as the number of cigarettes consumed per day. Eventually, the individual will be consuming tobacco products with essentially no nicotine. Since the nicotine-free tobacco products of the final step are non-addictive, it should then be much easier to quit the use of the tobacco products altogether.

EXAMPLE 22 Nicotine Reduction and/or Smoking Cessation Method Using Reduced Nicotine Cigarettes

The effectiveness of Quest® cigarettes was evaluated in a limited study. The chemical and physical characteristics of Quest® cigarettes are found in TABLES 3-10 (Examples 20 and 23). The primary objective of this prospective randomized controlled clinical trial was a continuous four-week period of abstinence from smoking. The study consisted of three treatment arms, comprised of 15 subjects each:

-   1) Quest 3®+nicotine patch -   2) Quest 3®+placebo patch -   3) Quest 1®, Quest 2® and Quest 3®.

The study was 18 weeks in duration. During the first week of the study subjects smoked only their usual brand of cigarette. From Weeks 2 through 5, Quest® cigarettes were introduced while subjects gradually reduced the amount of usual brand smoked (Groups 1 and 2) or the amount of nicotine per cigarette (Group 3). By Week 6, subjects were expected to be smoking Quest 3® cigarettes exclusively. From Weeks 6 through 11, subjects acclimated themselves to nicotine-free smoking. They were gradually weaned off Quest 3® (with phased discontinuation of nicotine patches for Groups 1 and 2) from Week 12 through Week 14 in anticipation of the four-week abstinence period during Weeks 15 through 18. Secondary endpoints evaluated included compensatory smoking behavior and withdrawal symptoms.

The overall success rate in achieving abstinence was 16 percent (7/45). Group 2 had the highest success, with approximately 33 percent of subjects (5/15) achieving four-week continuous abstinence. Smokers in Group 3 attained a 13 percent (2/15) abstinence rate. Group 1 experienced the lowest rates of success, with no subjects reaching the primary abstinence outcome. In addition, subjects who complied with the protocol regimen of use of nicotine-free Quest 3® cigarettes exclusively in the weeks leading up to their quit-smoking date had higher abstinence success rates (54 percent, 7/13). The failure of Group 1 (0 percent quit rate) was perceived as a methodology issue, and it was suggested that future studies incorporating a similar combination therapy have subjects wear the patch beyond the quit date.

Subjects rated Quest® cigarettes as less satisfying than their usual brand and, therefore, did not crave them as much as their regular brand of cigarette. This outcome was perceived by the author as beneficial since the lack of a craving for Quest® cigarettes may aid smoking cessation and also deter sustained use. Even though Quest® was less satisfying, subjects did not compensate for the lack of nicotine by smoking more cigarettes or inhaling more smoke in each puff. Across the three groups, the number of cigarettes smoked and the expired CO levels did not increase during the six weeks that subjects had free access to Quest® cigarettes.

The results of this pilot study indicate that Quest® cigarettes, especially in the format of decreasing levels of nicotine such as that which exists in Quest® 1,2,3, have the potential to be efficacious as a smoking cessation aid.

Jed Rose and F M Behm also conducted a blinded, randomized study designed to determine if compensatory smoking behaviors differ depending on whether highly ventilated filtered or reduced-nicotine filtered cigarettes are being consumed by the same subjects. Rose J E, Behm F M. “Effects of low nicotine content cigarettes on smoke intake” Nicotine and Tobacco Research 6:1-11(2004a) (see below).

A total of 16 smokers were enrolled in this study. The study consisted of two separate 8-hour sessions where subjects randomly smoked either low-nicotine filtered or highly ventilated filter cigarettes ad libitum during each session, with a counterbalanced order of smoking conditions presented across subjects. The standardized smoke yield of nicotine was 0.02 mg/cigarette for the Quest® brand low-nicotine cigarettes, and 0.2 mg/cigarette for the highly ventilated filter cigarettes. Endpoints evaluated included expired carbon monoxide, puff volume and number of puffs, craving, arousal, reward, and airway sensations.

The number of cigarettes smoked during each 8-hour session was significantly higher when subjects consumed the highly ventilated filter cigarettes (mean=11.9) versus the low-nicotine filtered cigarettes (mean=10.4). Cumulative puff volume taken from the last cigarette of the session (for which puffing topography was monitored) was also significantly higher for the highly ventilated filtered cigarettes (800 cc) than for the low-nicotine filtered cigarettes (500 cc). Subjects tended to take more puffs from the highly ventilated filtered cigarettes, though the difference was not statistically significant.

The investigators devised a scale to evaluate expired-air carbon monoxide, which they called the “compensation index.” Expired-air CO values, as measured at the end of each 8-hour session, were divided by the FTC standardized values for CO delivery for each type of cigarette. Thus, a higher ratio would indicate a compensatory increase in smoking behavior. Even though expired CO levels were higher for the low-nicotine cigarettes (due to a higher smoke yield), the compensation index was significantly higher for the highly ventilated filter cigarettes (index=11.1) than for the low-nicotine filtered cigarettes (index=2.4). (The compensation index was graphed incorrectly in the paper and an erratum reflecting the corrected values was submitted.)

The authors concluded that compensatory smoking behaviors increased significantly during the highly ventilated filter cigarette sessions, relative to the low-nicotine filtered cigarettes. While the study provided some evidence that smokers would be unlikely to compensate when switching from their usual brand of cigarette to low-nicotine cigarettes, these suggestions are indirect and uncontrolled since there was no session involving smokers' usual brand of cigarette. However, baseline CO readings obtained when subjects were smoking their usual brands of cigarettes were similar to the readings obtained when smoking Quests. The data obtained from these studies and others to date provide strong evidence that tobacco-use cessation programs that employ Quest 1®, Quest 2® and Quest 3® with or without NRT (e.g., the nicotine patch) are effective approaches to achieve cessation of tobacco use.

EXAMPLE 23 A Prospective, Double Blind, Randomized, Active-Controlled, Parallel Group, Multicenter Test to Evaluate the Effectiveness of Reduced Nicotine Cigarettes Alone and in Combination with Nicotine Replacement Therapy as a Smoking Cessation Aid

This Phase II study will assess the magnitude of effectiveness that can be obtained with Quest 1®, Quest 2® and Quest 3® alone and in combination with NRT compared to conventional pharmacologic therapy, i.e., the NRT patch alone, as a smoking cessation aid.

This will be a prospective, randomized, multicenter, double-blind, parallel, active controlled Phase II trial in which a total of 345 healthy smokers who are motivated to quit smoking will be enrolled and followed over 8 months at approximately 5 sites. Healthy smokers with a desire to quit smoking will be screened for eligibility. This screening will include documentation of relevant medical/smoking history including documentation of current usual brand, physical examination, urine pregnancy test (in women of child-bearing potential), drug screen, exhaled carbon monoxide (CO), saliva cotinine level and the completion of four questionnaires. Following screening, eligible subjects will be randomized in a 1:1:1 ratio to one of the three treatment arms:

Group 1: Quest 1®, Quest 2® and Quest 3® plus NRT (patch)

Group 2: Quest 1®, Quest 2® and Quest 3® plus placebo patch

Group 3: Active Control cigarettes (conventional) plus NRT (patch)

Quest Smoking Cessation Product is a tobacco-based (botanical) medical product in cigarette form. As a ‘finished good’, the product has been developed to meet specifications similar to conventional cigarettes in both design and use, as illustrated by the following product specifications. TABLE 10 QUEST ® CHEMICAL AND PHYSICAL SPECIFICATIONS Quest 1 ® Quest 2 ® Quest 3 ® Chemical: Tar (mg/cigt)  9.0 ± 1.0  8.0 ± 1.25  8.5 ± 1.0 Nicotine (mg/cigt)  0.59 ± 0.06  0.30 ± 0.05 <0.05 Moisture 12.4% ± 1.0% 12.4% ± 1.0% 12.4% ± 1.0% Carbon Monoxide 12.5 max 12.5 max 12.5 max (mg/cigt) Physical: Total Weight (mg/cigt) 984 ± 70 984 ± 70 943 ± 70 Circumference 24.6 ± 0.1 24.6 ± 0.1 24.6 ± 0.1 (mm) Pressure Drop 115 ± 10 115 ± 10 123 ± 10 (mm H₂O) Hardness (%), 74 ± 3 74 ± 3 74 ± 3 unequlilibrated

The genetically modified plant used to produce the low nicotine yielding tobacco product has been designated Vector 21-41. This line is a transgenic tobacco that produces very low nicotine levels by disrupting the normal expression of a quinolinic acid, phosphoribosyltransferase, a key enzyme in the biosynthetic pathway leading to the production of nicotine. Vector 21-41 tobacco is a comparable plant to traditional wild-type tobacco and more significantly, its non-transgenic parent, Burley 21 LA. Vector 21-41 does not express any novel agronomic trait such as insect resistance or herbicide tolerance.

The nicotine patch is an over-the-counter (OTC) product; brand names include Nicoderm CQ® and Nicotrol® and generic products are available as well. The protocol specifies compliance with current labeling for OTC patches. Careful consideration has been made to limit active patch exposure to 10 weeks with no more than 6 weeks of exposure to the highest dosage form (21 mg) and no more than 2 weeks exposure at lower weaning dosages of 14 mg and 7 mg respectively. The patch will be replaced daily (every 24 hours) as directed. In order to ensure blinding, a placebo patch will be utilized in this study as well.

The Active Control cigarette to be used by Group 3 is a conventional, American-blended cigarette with a tar content of 10.2±0.5 mg/cigarette and a nicotine content of 0.80±0.10 mg/cigarette, however, the cigarettes are “'sham-faded” (i.e., the nicotine content of the Active Control does not decrease, but it is administered in accordance with that of the Quest® Smoking Cessation Product to simulate the fading) every two weeks in parallel with the fading provided by Quest 1®, Quest 2® and Quest 3® in Groups 1 and 2.

The objective of the Active Control is to deliver a constant nicotine concentration with the same appearance and taste as the investigational product. This matching reduces confounding and/or bias in the trial outcome 1) that may be introduced by other features of the device that differ from the Active Control; or 2) that may lead to breaking of the blinding.

All participating subjects are exposed to 6 weeks of smoking (either Quest 1®, Quest 2®, Quest 3® or conventional “faded” cigarettes) and 12 weeks of application of the nicotine and/or placebo patch. The last 2 weeks of smoking and first 2 weeks of patch use overlap (Week 5 and 6).

Subjects in Group 1 and 2 will transition from their usual brand (UB) cigarettes to Quest cigarettes. During the first 6 weeks (Week 1-Week 6), subjects in Groups 1 and 2 will smoke ad libitum for two weeks at each Quest® nicotine level beginning with Quest 1®, then proceeding to Quest 2® and then to Quest 3®. At Week 5, Quest® use will be supplemented in the first treatment group with the addition of a transdermal nicotine patch (21 mg), while subjects in Group 2 will add a placebo patch. Visit 5 is considered the ‘quit date’ (quit all smoking).

Subjects in Group 1 will receive the 21 mg transdermal nicotine patch for an additional 4 weeks (total period Week 5-Week 10), followed by the 14 mg transdermal nicotine patch for 2 weeks (Week 11 and 12), the 7 mg transdermal nicotine patch for 2 weeks (Week 13 and 14) and a placebo patch for 2 weeks (Week 15 and 16). Subjects in Group 2 will receive a placebo patch only starting at Week 5 through Week 16.

The third treatment group is a blinded control for the exposure to Quest 1®, Quest 2® and Quest 3® in Group 1, whereby subjects are provided with an Active Control cigarette (nicotine dose comparable to a conventional, American-blended cigarette), but the cigarettes are “sham-faded” every 2 weeks in parallel with the fading provided by Quest 1®, Quest 2® and Quest 3® in Group 1. The NRT treatment for Group 3 is the current standard of care, indicating the addition of a placebo patch during Week 5 and 6 preceding the “quit date” at Week 7 (visit 5). Subjects in Group 3 will use the 21 mg transdermal nicotine patch for 6 weeks (Week 7-Week 12), followed by the 14 mg transdermal nicotine patch for 2 weeks (Week 13 and 14), and the 7 mg transdermal nicotine patch for 2 weeks (Week 15 and 16).

All patches, placebo and NRT, are 24-hour applications, and should be re-applied daily. Additionally, all subjects will receive behavioral support through a 10 minute individual counseling session at visit 1 by a certified smoking cessation counselor and printed materials (You Can Quit Smoking).

As described, Quest® use will be supplemented in the first treatment arm with the addition of a transdermal nicotine patch (21 mg) at the beginning of Week 5. The application of the patch 2 weeks prior to the quit date is intentional and has been included because this regimen has been associated with improved durability of long-term abstinence rates. Following the quit date, subjects will remain on a traditional patch schedule until the end of Week 14. The current design incorporates 2 weeks of placebo patch treatment following standard NRT treatment in order to promote consistency when assigning abstinence rates, maintain blinding and avoid within-trial study bias.

Schuurmans and colleagues (Schuurmans et al. 2004) found that nicotine patch pre-treatment before cessation increased sustained abstinence rates at 6 months. Overall sustained abstinence was observed in 17 percent of subjects at 6 months; 22 percent in an experimental group verses 12 percent in a placebo-controlled group. Historically, one would begin nicotine replacement therapy on his/her quit day confronting the subject not only with a sudden behavioral change, but also with a new route of receiving nicotine. Pre-treatment with NRT may weaken the association between nicotine intake (rewarding effect) and the behavior of smoking thereby aiding in a disassociation of the conditioned behavior pattern. Secondly, pre-patch treatment may reduce the need for inhaled nicotine. Finally, prior patch treatment may ease the transition from inhaled nicotine to transdermal nicotine replacement by familiarization with the mode of delivery. Similar support for patch pre-treatment has been suggested by Rose et al. (unpublished) who found that pre-treatment with a nicotine patch two weeks before the quit date resulted in 50 percent abstinence rates at 6 weeks.

Patch pre-treatment has been incorporated into the current study design as a means to further test its potential benefit. Smoking Quest 3® cigarettes may hold a particular advantage when paired with patch pre-treatment since Quest 3® contains no nicotine. Thus, smoking nicotine-free cigarettes during pre-treatment may allow for an even easier transition to the patch with subjects acclimating to their new nicotine source while still afforded the behavioral and sensory aspects provided by the cigarette.

The control group, Group 3, will comply with current labeling requirements for OTC patches indicating that treatment with active NRT will start following the quit date. Furthermore, careful consideration was made to limit active patch exposure to 10 weeks with no more than 6 weeks of exposure to the highest dosage form (21 mg) and no more than 2 weeks exposure at lower weaning dosages of 14 mg and 7 mg respectively.

The primary endpoint, i.e., 4 weeks of continuous abstinence (Weeks 7-10), will be assessed after treatment termination, thus at Week 11 (Visit 7). Abstinence rates will be verified by self reports and exhaled CO<10 ppm for each subject.

Quit rates at 3 and 6 months (at Visits 11 and 12) following treatment termination will be verified by self reports and exhaled CO<10 ppm.

Preference and satisfaction of Quests over usual brand will be determined by subjective questionnaires.

The severity of withdrawal symptoms including negative mood, urges to smoke and difficulty concentrating will be determined by subjective questionnaires.

Compensatory smoking will be determined by the number of cigarettes smoked and chemically verified by CO levels.

Three hundred forty-five (345) smokers who have provided informed consent for participation in this trial, who are capable of comprehending the nature of the study and who are likely to comply with the visit schedule are to be entered into the study provided they conform to the following criteria: Subjects must be considered by the investigator to be in general good health between 21 and 65 years of age; Subjects must have a history of smoking an average of 15 cigarettes or more per day for at least one year; Subjects must be motivated to quit smoking; Subjects must have a corrected CO measurement of greater than 15 ppm at baseline; Subjects must be willing and able to return for scheduled follow-up examinations for a total of 8 months; Subjects must read, understand, complete and be given a copy of the signed Informed Consent form; Subjects must be able to read, understand and complete the questionnaires independently.

Subjects will be excluded if they meet any of the following criteria: using any form of NRT or other tobacco-based product (chew, snuff, etc.); usual brand consists of a menthol variety; known serious pathophysiology or topical or systemic disorders of any kind that would confound the results of the study; Subjects who are using illegal drugs at baseline; pregnant or lactating, or who plan to become pregnant or lactating during the course of the study; participating in any other clinical trial of an investigational drug or device during the time of this clinical investigation or within 30 days prior to screening visit; taking anti-depressants, anti-psychotics, Clonidine, Zyban, or any of the classes of drugs listed in the Accutest 10-Multidrug screen; have a positive Accutest 10-Multidrug screen; systolic blood pressure over 140 mmHg and/or diastolic blood pressure over 90 mmHg; consume an average of 3 or more drinks of alcohol per day; consume greater than an average of 3 packs of cigarettes per day.

All study products will bear the following: “Caution: New Drug—Limited by Federal Law to investigational use by qualified investigators only. Keep out of the reach of children. For clinical trial use only.” Additionally, cigarette packs will communicate the following Surgeon General's warning:

“SURGEON GENERAL'S WARNING: Quitting Smoking Now Greatly Reduces Serious Risks to Your Health.”

Quest 1®, Quest 2®, Quest 3® and the Active Control will be packaged 20 cigarettes per pack and 10 packs per carton and labeled in accordance with their study visit period:

Week 1 & 2

Week 3 & 4

Week 5 & 6

Quest 1®, Quest 2®, Quest 3® and the Active Control will be packaged in identical packages, such that there will be no visible markings, and they will be indistinguishable from conventional cigarettes. Each carton of cigarettes will have a label on the box. The label will contain protocol number, study period, the three-digit subject identification number (preprinted), and the new drug caution.

A date-coding stamp will be embossed into the bottom of each cigarette pack and carton which will distinguish each Quest® product from one another and from the Active Control. The date-coding system will consist of an arbitrary number to avoid bias and will be traceable to the lot number for each production run of investigational drug product.

Labeling of the NRT patch and placebo patch will be similar to the cigarettes. NRT patches in 21 mg, 14 mg and 7 mg will be repackaged. Placebo patches will be manufactured to match the size, appearance and texture of an active NRT.

NRT patches in 21 mg, 14 mg and 7 mg and placebo patches will be repackaged in an identical manner and in accordance with the randomization scheme, as described above for the cigarette packaging. A total of 20 patches will be packaged in one box. The placebo patch box will be labeled with a subject identification number and one of the following time periods: Week 5 & 6, Week 7 & 8, Week 9 & 10, Week 11 & 12, Week 13 & 14, or Week 15 & 16. The 21 mg NRT patch box will be labeled with a subject identification number and one of the following time periods: Week 5 & 6, Week 7 & 8. Week 9 & 10, or Week 11 & 12. The 14 mg NRT patch box will be labeled with a subject identification number and one of the following time periods: Week 11 & 12, or Week 13 & 14. The 7 mg NRT patch box will be labeled with a subject identification number and one of the following time periods: Week 13 & 14, or Week 15 & 16.

Each study kit will contain the appropriate number of cigarette cartons and patches for one study subject. The kit will be labeled with the protocol number subject identification number (preprinted), subject initials, and the new drug caution.

Healthy male and female smokers interested in quitting smoking will be recruited from the community. The investigative staff will explain the study purpose, procedures and subject responsibilities to the potential participant. The subject's willingness and ability to meet the follow-up requirements will be determined. When it has been established that the subject is likely to be eligible for participation, written informed consent will be obtained. The subject will sign and date the informed consent form, and the person explaining the consent will also sign and date the consent form. One copy of the informed consent form will be retained with the subject records and a signed copy will be provided to the subject.

Once the written consent has been provided, the subject will undergo complete screening procedures, consisting of relevant medical history, physical examination, urine pregnancy test (in women of child-bearing potential), drug screen, exhaled CO, saliva cotinine and the completion of four questionnaires.

Prior to the study, Subject Identification (Subject ID) numbers will be randomly assigned a treatment group (1, 2 or 3) in a 1:1:1 ratio by an independent statistician. Balanced blocks of equal size will be assigned to each study treatment. The study is double-blind indicating that the investigator and subject will be blinded to the treatment. Only the statistician responsible for developing the randomization key and those in charge of manufacturing and packaging the Test Articles will view the randomization scheme. The kits will be pre-packaged, display a Subject ID number and contain all cigarettes and patches to be dispensed over the course of the trial. The maximum number of subjects enrolled at any one site is 100.

Once subject eligibility has been confirmed, the subject will be scheduled to return to the site in 7 days. At that time they will be enrolled in the study, and the next sequential Subject ID number will be assigned. This number will consist of 5 digits in the following format: XX-XXX, the first 2 digits represent the pre-assigned site number and the final three digits will be taken from the next sequential kit available. For example, the first patient enrolled at site “02”, which had been allocated the next available kit number 180, would be assigned the Subject ID number 02-180. A subject who is screened, but not entered into the study (a screen fail) will be recorded on a screen log.

The Subject ID number and subject initials are to be recorded on all study documents and will link the study treatment and the study documents to the subject's name and (medical) record. To maintain confidentiality, the subject's name or any other personal identifiers should not be recorded on any study document other than the informed consent form.

Subjects will be examined and evaluated according to the following schedule of visits: Visit 1: Screening/Baseline (day—7 through day 0); Visit 2: Randomization (day 0) ±2 days post screening; Visit 3: 2 weeks±3 days post randomization, Visit 4: 4 weeks±3 days post randomization; Visit 5: 6 weeks±3 days post randomization (quit date); Visit 6: 8 weeks±3 days post randomization; Visit 7: 10 weeks±3 days post randomization; Visit 8: 12 weeks±3 days post randomization; Visit 9: 14 weeks±3 days post randomization; Visit 10: 16 weeks±3 days post randomization; and Visit 11: 18 weeks±7 days post randomization (3 month quit rate); Visit 12: 31 weeks±7 days post randomization (6 months quit rate).

The clinical parameters to be evaluated are: Self report diaries (visits 1 through 5); saliva cotinine concentration (all Visits); Exhaled, background, and corrected CO (all visits); blood pressure and heart rate (all visits); Minnesota smoking withdrawal questionnaire (all visits); Cigarette evaluation questionnaire (visits 1 through 5); Sensory questionnaire (visits 1 through 5); Fagerström Test of Nicotine Dependency (visits 1 through 5); and DNA blood analysis.

A Case Report Form (CRF) booklet will be provided by Vector Tobacco or its designee for each subject enrolled in the study. The appropriate Case Report Form will be completed at each visit. The investigator will sign each subject's completed CRF once he/she has reviewed all data contained within. All CRFs will be completed in a legible manner in black ink. Any corrections will be made by drawing a single line through the incorrect entry, entering the correct information and initialing and dating the change.

All clinical data generated in the study will be submitted to Vector Tobacco or its designee for quality assurance review, data entry, and statistical analysis. All forms will be reviewed for completeness and evident recording errors will be rectified by contact with the appropriate clinical site. Informed consent must be obtained prior to any study-specific procedure and use of the study product. A signed and dated informed consent form will be obtained from each subject in accordance with ICH GCPs and with local regulatory and legal requirements. The original, completed informed consent form must be retained by the investigator as part of the study records and a signed copy must be given to the subject. All study visits should take place after 12 pm.

The following procedures and observations will be performed at visit 1 (day—7 through day 0): signed written informed consent will be obtained; screening for inclusion and exclusion criteria will be completed; drug screen will be performed; demographic information will be collected; relevant medical history will be taken; smoking history (including number of cigarettes smoked per day, and documentation of current usual brand) will be taken; You Can Quit Smoking booklet will be dispensed; ten minute counseling session will be provided; concomitant medication history will be completed (for the first visit, this will need to state all medications taken within the previous 7 days); physical exam will be performed; urine pregnancy testing will be performed; blood pressure and heart rate will be determined; height will be measured; weight will be measured; diary card and instructions for completing it will be given (subjects will be smoking their usual brand of cigarettes during this week); saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; Smoking Withdrawal Questionnaire will be completed; Cigarette Evaluation Questionnaire will be completed (for usual brand); Sensory Questionnaire will be completed (for usual brand); Fagerström Test of Nicotine Dependency will be completed; and subjects will be scheduled to return to the site in 7 days (±2 Days) for visit 2.

The following procedures and observations will be performed at visit 2 (day 0): blood for DNA analysis will be drawn (if applicable); randomization assigned; confirmation of inclusion/exclusion criteria; concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; diary entries will be reviewed with subject, feedback provided and new diary cards dispensed; saliva will be analyzed for cotinine; Exhaled CO, background CO, and corrected CO concentrations will be measured; first 2 weeks of randomized treatment will be dispensed; Smoking Withdrawal Questionnaire will be completed; Cigarette Evaluation Questionnaire will be completed (for usual brand); Sensory Questionnaire will be completed (for usual brand); Fagerström Test of Nicotine Dependency will be completed; subjects will be instructed to bring their unused cigarette packs and diary to the site; serious adverse events will be reviewed and recorded; and subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 3.

The following procedures and observations will be performed at visit 3 (week 2): concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; diary card will be supplied to document cigarette usage; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; week 3 & 4 of assigned randomized treatment will be dispensed; Smoking Withdrawal Questionnaire will be completed; Cigarette Evaluation Questionnaire will be completed: Sensory Questionnaire will be completed; Fagerström Test of Nicotine Dependency will be completed; use will be determined by the number of returned cigarettes; adverse events will be reviewed and recorded; subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 4; and subjects will be reminded to bring their unused cigarette packs and diary to the site.

The following procedures and observations will be performed at visit 4 (week 4): concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; diary card will be supplied to document cigarette usage; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; week 5 & 6 of assigned randomized treatment (cigarettes and patches) will be dispensed; Smoking Withdrawal Questionnaire will be completed, Cigarette Evaluation Questionnaire will be completed; Sensory Questionnaire will be completed; Fagerström Test of Nicotine Dependency will be completed; use will be determined by the number of returned cigarettes; adverse events will be reviewed and recorded; subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 5; and subjects will be reminded to bring their unused cigarette packs, unused patches and diary to the site.

The following procedures and observations will be performed at visit 5 (week 6-quit date): the quit date, as of this visit, will be emphasized; complete concomitant medication history (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; saliva analysis for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; week 7 & 8 of assigned randomized treatment (patches only) will be dispensed; Smoking Withdrawal Questionnaire will be completed; Cigarette Evaluation Questionnaire will be completed; Sensory Questionnaire will be completed; Fagerström Test of Nicotine Dependency will be completed; use will be determined by the number of returned cigarettes; compliance will be determined by the number of returned patches; review and record adverse events; subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 6; and remind the subjects to bring their unused patches to the site.

The following procedures and observations will be performed: complete concomitant medication history (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; saliva analysis for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; week 9 & 10 of assigned randomized treatment (patches only) will be dispensed; smoking Withdrawal Questionnaire will be completed; compliance will be determined by the number of returned patches; all subjects will be asked if they have smoked since the last visit and if they have smoked within the last 7 days; review and record adverse events; subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 7; and remind the subjects to bring their unused patches to the site.

The following procedures and observations will be performed at visit 7 (week 10): concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; week 11 & 12 of assigned randomized treatment (patches only) will be dispensed; smoking Withdrawal Questionnaire will be completed; compliance will be determined by the number of returned patches; subjects will be asked if they have smoked since the last visit and if they have smoked within the last 7 days; adverse events will be reviewed and recorded; subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 8; and subjects will be reminded to bring their unused patches to the site.

The following procedures and observations will be performed at visit 8 (week 12): concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last Visit); blood pressure and heart rate will be determined; weight will be measured; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; week 13 & 14 of assigned randomized treatment (patches only) will be dispensed; smoking Withdrawal Questionnaire will be completed; compliance will be determined by the number of returned patches; subjects will be asked if they have smoked since the last visit and if they have smoked within the last 7 days; adverse events will be reviewed and recorded; subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 9; and subjects will be reminded to bring their unused patches to the site.

The following procedures and observations will be performed at visit 9: concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; week 15 & 16 of assigned randomized treatment (patches only) will be dispensed; smoking Withdrawal Questionnaire will be completed; compliance will be determined by the number of returned patches; subjects will be asked if they have smoked since the last visit and if they have smoked within the last 7 days; adverse events will be reviewed and recorded; subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 10; and subjects will be reminded to bring their unused patches to the site.

The following procedures and observations will be performed at visit 10 (week 16): concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); physical exam will be performed; Urine pregnancy testing will be performed; blood pressure and heart rate will be determined; weight will be measured; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; Smoking Withdrawal Questionnaire will be completed; compliance will be determined by the number of returned patches; subjects will be asked if they have smoked since the last visit and if they have smoked within the last 7 days; adverse events will be reviewed and recorded; and subjects will be scheduled to return to the site in two weeks (±3 Days) for visit 11.

The following procedures and observations will be performed at visit 11 (week 18): concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; smoking Withdrawal Questionnaire will be completed; subjects will be asked if they have smoked since the last visit and it they have smoked within the last 7 days; adverse events will be reviewed and recorded; and subjects will be notified that they will receive a follow-up phone call in approximately 13 weeks to determine their smoking status.

For visit 12 (week 31), the subject will be questioned regarding their smoking status by telephone. Subjects who indicate that they have relapsed within the past 7 days will be questioned about adverse events and terminated from the study. Subjects who indicate that they are still abstinent, or have smoked only since the last visit and not within the past 7 days will be asked to come to the site within one week of the phone call (±3 days) for the following procedures and observations: concomitant medication history will be completed (for this and all subsequent visits, this will need to state all medications taken since last visit); blood pressure and heart rate will be determined; weight will be measured; saliva will be analyzed for cotinine; exhaled CO, background CO, and corrected CO concentrations will be measured; alcohol intake will be assessed; Smoking Withdrawal Questionnaire will be completed; adverse events will be reviewed and recorded; and subjects will exit the study.

Subjects are considered to have completed the study if they have completed all follow-up examinations through visit 12.

Subjects may be terminated from the study at the discretion of the investigator only for reasons related to the study treatment regimen that would jeopardize the subjects' health and/or welfare if they were to continue in the study. Terminated subjects will be considered to have completed the study and should not be replaced. However, every effort will be made to follow terminated subjects (due to an adverse event) for safety reasons using the appropriate case report forms until the planned end of the study period. Notification of a subject termination due to an adverse event will be made immediately to Vector Tobacco or its designee.

Subjects may be discontinued from the study for non-treatment-related reasons only when no other option is possible. Reasons for discontinuation include, but are not necessarily limited to, 1) voluntary withdrawal from the study by the subject; 2) subject has moved from the area and is determined to be lost to follow-up; 3) subject is unwilling or unable to cooperate with study requirements (assigned treatment, follow-up visits, etc.). The reason for discontinuation will be recorded on the appropriate case report form. Discontinued subjects will not be replaced in the study.

Subjects who do not quit smoking after visit 5 quit date or start smoking again after the visit 5 quit date and prior to visit 10 will be discontinued from the study. All assessments for that specific visit should be performed including a physical and pregnancy test, if applicable.

Prior to discontinuing a subject, every effort should be made to contact the subject to either encourage the subject to maintain compliance with the protocol or to obtain as much follow-up data as possible regarding the subject's current status. Efforts to contact the subject will consist of documentation of at least three attempts to contact the subject by phone followed by at least two certified letters with return receipt. A study completion form must be completed for all subjects who complete, discontinue, or are terminated from the study.

EXAMPLE 24 A Nicotine Reduction and/or Smoking Cessation Method Using a Reduced Nicotine Tobacco Product in Conjunction with a Nicotine Patch

The efficacy of a nicotine reduction and/or smoking cessation program using both a reduced nicotine cigarette and a nicotine patch has been evaluated. The primary objective of this prospective randomized controlled clinical trial was a continuous four-week period of abstinence from smoking. The study consisted of a group of 15 subjects (one of three treatment arms in the overall study).

The study was 18 weeks in duration. During the first week of the study, subjects smoked only their usual brand of cigarette. From Weeks 2 through 5, Quest 3® cigarettes (having less than 0.05 mg nicotine) were introduced while subjects gradually reduced the amount of usual brand smoked. By Week 6, subjects were expected to be smoking Quest 3® cigarettes exclusively. From Weeks 6 through 11, subjects acclimated themselves to nicotine-free smoking. They were gradually weaned off Quest 3® with phased discontinuation of nicotine patches from Week 12 through Week 14 in anticipation of the four-week abstinence period during Weeks 15 through 18. Secondary endpoints evaluated included compensatory smoking behavior and withdrawal symptoms.

Subjects rated Quest® cigarettes as less satisfying than their usual brand and, therefore, did not crave them as much as their regular brand of cigarette. This outcome was perceived as beneficial since the lack of a craving for Quest® cigarettes may aid smoking cessation and also deter sustained use. Even though Quest® was less satisfying, subjects did not compensate for the lack of nicotine by smoking more cigarettes or inhaling more smoke in each puff. The number of cigarettes smoked and the expired CO levels did not increase during the six weeks that subjects had free access to Quest® cigarettes. The results of this study showed that Quest® cigarettes have the potential to be efficacious as a smoking cessation aid.

EXAMPLE 25 A Nicotine Reduction and/or Smoking Cessation Method Using a Reduced Nicotine Tobacco Product in Conjunction with a Nicotine Patch

In a study by Rose et al. (presented at the Society for Research in Nicotine and Tobacco 2005 Annual Meeting), fifteen smokers (6 males, 9 females; mean FTND score 6.9) participated in a study designed to investigate the neuroanatomical substrates of nicotine dependence. Dependence was manipulated by having subjects switch to smoking low nicotine content cigarettes while wearing nicotine skin patches; this manipulation, which reduces exposure to inhaled nicotine, has been shown to reduce indices of nicotine dependence. Participants were assessed using positron emission tomography (PET) to measure changes in regional cerebral metabolic rate for glucose (rCMRglc) and regional cerebral blood flow (rCBF). Subjects were scanned during three sessions conducted after overnight abstinence from smoking: 1) at baseline; 2) after two weeks of low nicotine content cigarettes (<0.1 mg nicotine delivery)+nicotine patches (21 mg/24 h, removed the night before test sessions); and 3) two weeks after returning to smoking their usual brand of cigarettes (mean nicotine delivery 0.8 mg). Craving for cigarettes decreased significantly at the second session (after 2 weeks exposure to low nicotine containing cigarettes+nicotine patches) relative to the first and last sessions (p=0.03). FTND score (assessed at each session) also decreased at the second session (p=0.06). The right hemisphere anterior cingulated cortex similarly showed a significant decrease in activation (based on rCMRglc measures) at the second session (p=0.002). These results confirmed previous findings that exposure to reduced nicotine content cigarettes plus nicotine patches can lead to a reduction in nicotine dependence, and offer additional support for the view that activation of the anterior cingulated cortex is a neural correlate of drug craving.

EXAMPLE 26 Nicotine Reduction and/or Smoking Cessation Kit Containing Packs of Cigarettes with Low TSNA Levels and Stepwise Reductions in Nicotine Levels

Various nicotine reduction and/or smoking cessation kits are prepared, geared to heavy, medium, or light smokers. The kits provide all of the materials needed to quit smoking in either a two-week period (fast), a one-month period (medium) or in a two-month period (slow), depending on the kit. Each kit contains a set number of packs of cigarettes modified according the present invention, containing either step 1 cigarettes containing 0.6 mg nicotine, step 2 cigarettes containing 0.3 mg nicotine, and step 3 cigarettes containing less than 0.05 mg nicotine. For example, 1 pack a day smokers would receive 7 packs of cigarettes, each pack containing the above amounts of nicotine per each cigarette. Several weeks worth of additional cigarettes containing less than 0.05 mg nicotine/cigarette would also be provided in the kit, to familiarize the consumer with smoking no nicotine cigarettes. The kit may also contain a diary for keeping track of daily nicotine intake, motivational literature to keep the individual interested in continuing the cessation program, health information on the benefits of smoking cessation, and web site addresses to find additional anti-smoking information, such as chat groups, meetings, newsletters, recent publications, and other pertinent links.

Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All references cited herein are hereby expressly incorporated by reference. 

1. A tobacco use cessation kit comprising: a first tobacco product that comprises nicotine and delivers a collective content of N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) of less than 5.0 μg/g; a second tobacco product that comprises an amount of nicotine that is less than the first tobacco product and delivers a collective content of N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) of less than 5.0 μg/g, wherein said first tobacco product and said second tobacco product are cigarettes; and a third tobacco product that comprises an amount of nicotine that is less than the amount of nicotine in said second tobacco product and delivers a collective content of N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) of less than 5.0 μg/g, wherein said first tobacco product, said second tobacco product, and said third tobacco product are cigarettes and wherein said first tobacco product delivers less than 0.7 mg/g nicotine, said second tobacco product delivers less than 0.4 mg/g nicotine, and said third tobacco product delivers less than 0.5 mg/g nicotine. 